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Multiple Myeloma (MM) remains a cancer of terminally-differentiated plasma cells that reside predominantly within the bone marrow. Malignant plasma cells are nurtured by a permissive microenvironment that favors tumor progression, drug resistance and disease relapse. Recurrent somatic mutations in hematopoietic stem cells, the source of major components in bone marrow niche, lead to age-associated clonal hematopoiesis of indetermined potential (CHIP) and has been associated with inferior survivals among individuals without malignant hematologic disorders. It is possible that these mutations in non-MM cells would affect remission time after transplant. We performed whole exome sequencing to identify CHIP-associated mutations within the autograft utilized to rescue hematopoiesis after high dose melphalan and autologous hematopoietic cell transplant (HCT). We then correlated the presence of CHIP with the outcome of MM patients (pts) following autologous HCT. Methods. MM pts (N=101) that underwent HCT between 2006 and 2017 were studied. DNA was extracted from 1 ml of cryopreserved mobilized hematopoietic cell product. Targeted sequencing was then performed using Tempus xE whole exome platform (Tempus, Chicago, IL) with variable allele frequency (VAF) ≥0.1. Progression-free survival (PFS) and overall survival (OS) were defined as the time from transplantation until event of interest, with censoring at time of last follow up. Cox regression was used for time-to-event outcomes; hazard ratios (HR) and 95% confidence intervals (CI) were reported. P-values were two-sided and those Results. The median age was 60.1 years at the time of HCT. 58 pts were male and 43 female; 29 pts were African-American, 1 Asian/Indian and 71 were Caucasian. Seventy five (75%) and 42 (43%) pts had prior exposure to lenalidomide or cyclophosphamide, respectively. Thirty two pts (32%) underwent hematopoietic cell mobilization with GCS-F and plerixafor, 38 pts (38%) with cyclophosphamide and 31 pts (31%) with GCS-F alone. Forty seven pts (47%) had at least one CHIP-associated mutation. The median number of CD34+ yield in the first day was 6.7 x 10e6/kg for pts with CHIP mutations and 5.64 x 10e6/kg for pts without any CHIP mutation (p = 0.4). The presence or absence of CHIP mutations did not impact the yield of CD34+ stem cells collected (Fig. 1). The majority of mutations were either missense variants or mRNA splicing variations (Fig. 2), most commonly SF3B1, ASXL1, TET2 and ASXL2. CHIP mutations were associated with increased age (p= 0.021), but there was no association with prior history of previous cancer, smoking, cytogenetically-defined high risk disease, pre-HCT hemoglobin level or red blood cell mean corpuscular volume. There was also no significant difference between the transplant course between the two groups in terms of neutrophil and platelet engraftment or length of hospital stay. Median follow up was 32 months. During follow up 3 pts develop MDS/AML (2 pts in the CHIP group and one in no CHIP group). Coronary artery disease was the cause of death in one patient in CHIP cohort versus none in the CHIP negative cohort. Myeloma progression was the major cause of death in CHIP and No CHIP cohorts (66% vs. 60%, p=0.48). Median PFS was 19 months in CHIP cohort vs. 39 months in No CHIP group (HR: 0.62, 95% CI: 0.51-0.92, p= 0.001). Median OS has not been reached in the No CHIP group while it was 56 months in CHIP group (HR: 0.78, 95% CI: 0.62-1.04, P=0.09). In multivariate analysis, presence of CHIP remained a significant predictor of worse PFS after adjusting for age, maintenance therapy, high risk disease, performance status, triplet vs. doublet pre-SCT therapy, melphalan dose and MM genetic risk. (Table 2). Conclusion: Here we showed high frequency of large clonal hematopoiesis clones (i.e., VAF ≥0.1) in autograft of MM pts. The presence of this mutations does not impact stem cell yield or transplant course, however it impacts PFS independent of age and other unfavorable factors. Our data highlights high frequency of SF3B1; the role of this mutant in modulating apoptotic factors in myeloma microenvironment should be investigated. Disclosures Malek: Celgene: Consultancy; Amgen: Speakers Bureau; Adaptive: Consultancy; Janssen: Speakers Bureau; Medpacto: Research Funding; Sanofi: Consultancy; Takeda: Consultancy. Caimi:ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Fate Therapeutics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Caldwell:Tempus Inc.: Employment.

Amyloid light-chain (AL) amyloidosis results from extracellular deposition of fibril-forming monoclonal immunoglobulin light chains (LCs) usually secreted by a plasma cell (PC) clone. Misfolded LCs deposit as unique fibrils leading to organ failure and eventually death. Survival for untreated patients remains poor, and restrictive cardiomyopathy, nephrotic syndrome or hepatic failure greatly worsen outcome. Conventional chemotherapy and novel therapy with immunomodulatory drugs or proteasome inhibitors (PIs) eradicate PCs but generate adverse toxicities, and sustained responses are limited. Moreover, only 20% of patients with AL amyloidosis are eligible for stem cell transplant. The molecular events deregulated in AL amyloidosis remain undefined, and effective therapies based upon the biology of the disease are lacking. Impressive hematologic response rates obtained with bortezomib provide practice-changing data to advocate their introduction early in the treatment course. Bortezomib and the emerging second-generation PIs alone or combined with dexamethasone and alkylating agents may enhance hematologic and organ responses in AL amyloidosis.

Theragnostics represent cutting-edge, multi-disciplinary strategies that combine diagnostics with therapeutics in order to generate personalized therapies that improve patient outcome. In oncology, the approach is aimed at more accurate diagnosis of cancer, optimization of patient selection to identify those most likely to benefit from a specific therapy and to generate effective therapeutics that enhance patient survival. MicroRNAs (miRNAs) are master regulators of the human genome that orchestrate myriad cellular pathways to control growth during physiologic and pathologic conditions. Compelling evidence shows that miRNA deregulation promotes events linked to tumor initiation, metastasis and drug resistance as seen in multiple myeloma (MM), an invariably fatal hematologic malignancy. miRNAs are readily detected in body fluids, for example, serum, plasma, urine, as well as circulating tumor cells to demonstrate their potential as readily accessible, non-invasive diagnostic and prognostic biomarkers and potential therapeutics. Specific miRNAs are aberrantly expressed early in myelomagenesis and may more readily detect high-risk disease than current methods. Although only recently discovered miRNAs have rapidly advanced from preclinical studies to evaluation in human clinical trials. The development of miRNA theragnostics should provide widely applicable tools for the targeted delivery of personalized medicines to improve the outcome of patients with MM.

While the clinical benefit of proteasome inhibitors (PIs) for multiple myeloma (MM) treatment remains unchallenged, dose‐limiting toxicities and the inevitable emergence of drug resistance limit their long‐term utility. Disease eradication is compromised by drug resistance that is either present de novo or therapy‐induced, which accounts for the majority of tumor relapses and MM‐related deaths. Non‐coding RNAs (ncRNAs) are a broad class of RNA molecules, including long non‐coding RNAs (lncRNAs), that do not encode proteins but play a major role in regulating the fundamental cellular processes that control cancer initiation, metastasis, and therapeutic resistance. While lncRNAs have recently attracted significant attention as therapeutic targets to potentially improve cancer treatment, identification of lncRNAs that are deregulated in cells resistant to PIs has not been previously addressed. We have modeled drug resistance by generating three MM cell lines with acquired resistance to either bortezomib, carfilzomib, or ixazomib. Genome‐wide profiling identified lncRNAs that were significantly deregulated in all three PIresistant cell lines relative to the drug‐sensitive parental cell line. Strikingly, certain lncRNAs deregulated in the three PI‐resistant cell lines were also deregulated in MM plasma cells isolated from newly diagnosed patients compared to healthy plasma cells. Taken together, these preliminary studies strongly suggest that lncRNAs represent potential therapeutic targets to prevent or overcome drug resistance. More investigations are ongoing to expand these initial studies in a greater number of MM patients to better define lncRNAs signatures that contribute to PI resistance in MM.

Amyloidoses represent a highly heterogeneous group of diseases characterized by the abnormal production and accumulation of abnormal, insoluble amyloid proteins in various tissues leading to organ dysfunction. Light‐chain (AL) amyloidosis is the most common form of systemic amyloidosis and is characterized by extracellular deposi‐ tion of pathologic insoluble fibrillar proteins in organs and tissues. Primary systemic AL amyloidosis (AL) arises from the production of abnormal immunoglobulins (Igs) by clonal plasma cells, such as those associated with the plasma cell dyscrasia multiple myeloma (MM). AL amyloidosis can affect a wide range of organs, most commonly the kidneys, and consequently presents with a range of symptoms. Currently, the most effective treatment is autologous bone marrow transplants with stem cell rescue, but many patients are too weak to tolerate this approach and are ineligible. Novel therapeutic strategies recently used include forms of chemotherapy and targeted therapy similar to those used to treat MM. As a single agent, the proteasome inhibitor bortezomib has notable activity in selected populations of patients with relapsed AL. Here, we discuss recent advances using proteasome inhibitors to improve the outcome of AL amyloidosis patients.

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Targeting the ubiquitin+proteasome protein degradation pathway with the therapeutic agent bortezomib has significantly improved the survival of cancer patients but drug resistance inevitably develops. Aggresomes and the autophagy pathway serve as compensatory protein-clearance mechanisms that eradicate potentially toxic proteins to promote resistance to proteasome inhibitors and, hence, tumor survival. Pre-clinical evidence has emerged to demonstrate active crosstalk between these protein degradation pathways and has revealed novel therapeutic targets and strategies. Translational research and clinical trials are now focused on these pathways to prevent the emergence of drug resistance, enhance apoptosis and further improve the survival of cancer patients.

The ubiquitin+proteasome system (UPS) is a highly complex network that maintains protein homeostasis and cell viability through the selective turnover of targeted substrates. The proteasome serves as the catalytic core of the UPS to recognize and execute the coordinated and efficient removal of ubiquitinated proteins. Pharmacologic inhibitors that exploit the pivotal role of the proteasome in cellular metabolism promote tumor cytotoxicity and have yielded durable clinical responses that dramatically improve patient survival. Success of the proteasome inhibitor (PI) bortezomib in the treatment of the hematologic malignancy multiple myeloma (MM) has emerged as the standard-of-care and catapulted the UPS into a position of prominence as a model system in cancer biology and drug development. However, expansion of PIs in the treatment of the more complex solid tumors has been less successful. While clinical evaluation of second-generation PIs progresses, other potential sites of therapeutic intervention within the UPS continue to emerge, such as the non-proteolytic activities associated with the proteasome and the rapidly expanding number of Ub-binding proteins. Molecular-genetic approaches to further unravel the complexity of the UPS will advance its utilization as a platform for the development of novel, mechanism-based anticancer strategies.

Multiple myeloma (MM) is characterized by the loss of critical mediators of immune surveillance and the activation of osteoclast. The transforming growth factor-beta (TGF-β) signaling pathway has been implicated in MM progression through promotion of catabolic bone remodeling, IL-6 secretion and Th17 T cell development, promoting osteolytic bone disease, immune suppression and MM progression. Here, we conducted preclinical studies in the syngeneic 5T3MM immunocompetent mouse model of MM to assess the single agent activity of vactosertib (Vacto, TEW-7197), a TGF-β type I receptor kinase inhibitor, and the potential to act synergistically with the third-generation immunomodulatory drug, pomalidomide (Pom). These data support the first clinic trial to evaluate this combination regimen in patients (pts) with MM (NCT03143985, open and recruiting). Methods Preclinical: Mice bearing 5T33MM cells expressing luciferase were treated with Vacto, Pom and the combination for 3 weeks, and evaluated for MM growth by bioluminescence imaging (BLI). Cellular and molecular assays were performed in human RPMI8226 and U266 as well as murine 5T33MM cells via apoptosis, real-time PCR and western blotting. Peripheral blood monoclonal protein concentration, M-spike, was measured by ELISA. Distal femur trabecular bone structure was assessed by 3D micro-CT. Phase I clinical trial: In this open-label study, pts with relapsed MM with at least two lines of therapies received escalating dosages of Vacto in combination with a standard dose of Pom (4 mg) without corticosteroids. The study was conducted as a modified Fibonacci 3 + 3 dose escalation design to assess safety, maximum tolerated dose, recommended phase 2 dose, and efficacy of Vacto/Pom combination in compared to historical control of Pom without corticosteroids (PFS-6: 20% in randomized Phase II study by Richardson et al. Blood. 2014). Vacto tablets are administered orally in 28-day cycles, taken once daily for 5 days followed by 2 days without treatment, repeated either for 6 cycles or until progression of disease or intolerable toxicity. Dosing was initiated at 60 mg and increased to 120 and 240 mg. Results Preclinical: Vacto attenuated the growth and viability of human and murine MM cells by inducing apoptosis and inhibited TGF-β-induced activation of Smad2/3 in MM cells in vitro. In the 5T33MM preclinical model, single agent Vacto inhibited MM progression as measured by peripheral blood monoclonal protein concentration and BLI. Vacto also prolonged survival (Figure-1), prevented weight loss and increased trabecular bone thickness (Figure-2) in mice bearing MM. Vacto either alone or in combination with Pom also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b⁺Gr-1⁺ myeloid derived suppressor cells (MDSCs) in the bone marrow and diminished the population of Foxp3⁺ regulatory T cells in the spleen (Figure-1). Vacto showed anti-myeloma activity in cells from three MM pts (Figure-3) at 700-750 nM in vitro. Phase I: As of 01 July 2018, 5 pts were enrolled; 3 pts received 60 mg daily and 2 pts received 120 mg daily. Patient demographics include: median age of 70 years (range: 64-77); 3-5 lines of prior therapy; ISS stage II or III; two pts had high risk cytogenetics including 17p deletion; three pts were refractory to bortezomib and four pts were refractory to lenalidomide. None of five enrolled pts developed any DLT or needed Vacto dose reduction .The most non-hematologic common adverse event (AE) was grade II fatigue and pain in one pt with no grade III or IV non-hematologic AEs. No pt experienced any progression of disease (PFS-6: 100%). Figure-4 illustrates fluctuations in disease markers at each cycle interval. Two pts had grade III hematologic AE, no grad IV hematologic AE. Conclusions: Vacto has activity against myeloma in the syngeneic 5T33MM murine MM mouse model and in both human MM cell lines and primary patient MM samples. Remarkably, the effect of Vacto on the tumor microenvironment is augmented in combination with Pom in the Cereblon-negative immunocompetent 5T33MM model. The phase I data show safety of this agent in combination with Pom. The preliminary efficacy assessment is PFS-6: 100% which is higher than historical controls (Richardson et al. Blood. 2014) with Pom only (PFS-6: 20%) or Pom with corticosteroids (PFS-6:40%). Advancement of Vacto in clinical trial pipelines for MM and other malignant hematological disorders is planned. Disclosures Malek: Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau. Caimi:Kite Pharma: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees. Kim:MedPacto: Other: Founder/CEO.

Lenalidomide maintenance therapy, initiated around day 100 after autologous stem cell transplantation (ASCT), has been associated a significantly longer time to disease progression and significantly improved overall survival (OS) among patients with multiple myeloma (MM) (McCarthy et al. NEJM 2012). However, the impact of maintenance therapy on the nature of MM relapse is largely unknown. Generally relapse can be categorized in two broad categories as symptomatic, or biochemical relapse. Symptomatic relapse is characterized mostly by high calcium, renal failure, anemia and bony lesions, i.e., CRAB criteria. Biochemical relapse is defined by a resurgence of secretory function of malignant plasma cells without CRAB criteria. We hypothesized that long-term continuous exposure to lenalidomide induces crucial changes in the myeloma microenvironment leading to a discordance between the resurgence of secretory function and the recurrence of aggressive behavior of myeloma cells leading to a higher frequency of biochemical. To define the impact of maintenance therapy on the pattern of disease recurrence and determinants of a more aggressive or indolent relapse course, we retrospectively analyzed two cohorts of MM patients that underwent ASCT. Methods: All MM patients with measurable disease who received ASCT at the University Hospital, Cleveland, OH from 2007 to 2016 were reviewed (Fig. 1). Patients that had received an allogeneic transplant or tandem ASCT, those transplanted more than one year after induction therapy, and those that died during the first three months post-transplantation were excluded. Baseline and treatment characteristics, response status, and monitoring data prior to relapse were extracted. If the serologic relapse had coincided with clinical relapse, defined as per CRAB criteria, patient was categorized as clinical relapse, and otherwise as biochemical relapse. Type of MM was categorized as paraprotein only (PO), paraprotein and light chain (PL), light chain escape (LE) and clinical only (CO) relapse (Table 1). Baseline and treatment characteristics are detailed in Table 2. The univariate association between the type of relapse and key patient and clinical characteristics were assessed using t-test or Fisher's exact test as appropriate. Results: Study population included 124 pts who relapsed after ASCT (Fig. 1), divided in two cohorts based on history of receiving any maintenance therapy or no maintenance (77 vs. 47 patients, respectively). 21 pts were excluded based on the above criteria. Median age was 59.8 years old (range: 36-79). The median time from diagnosis to ASCT was 10 months. There was only one CO relapse in the maintenance cohort. There was no difference in rate of high risk disease between two cohorts. The maintenance agents included immunomodulatory drugs in 70 pts (91%), proteasome inhibitors in 5 pts (6%) and a combination of both in 2 pts (3%). PFS was 40.1 months in the maintenance group vs. 29.2 months in the no maintenance group (p=0.0001). There was no statistically significant difference in serum paraprotein values at diagnosis or relapse between the two cohorts (Fig. 1A). LCE was more common in the maintenance group. Thirteen patients (28%) in the no maintenance cohort had CRAB relapse compared to 35 patients (45%) in the maintenance cohort (hazard ratio: 0.71, 95% CI: 0.51-0.92, p=0.031). The type of end organ dysfunction at clinical relapse is shown in bar graphs in Fig. 2B. Conclusions: Taken together, our results suggest that although maintenance therapy postpones disease relapse in the post-HSCT setting, it also impacts dynamics of the resurgence of secretory function within myeloma cells and shifts the nature of recurrence toward less biochemical relapse. Future studies will employ next generation sequencing prior to and during maintenance therapy combined with high sensitivity flow cytometry to identify the molecular basis of relapse. Genomics and cell surface markers may then more accurately predict relapse in MM patients on maintenance therapy. Disclosures Caimi: Genentech: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau. Malek:Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Multiple Myeloma (MM) are often treated with high-dose melphalan and autologous stem cell transplant (ASCT), followed by maintenance therapy (McCarthy et al. JCO, 2017). Cardiac toxicity is a potentially serious complication and is a dose limiting toxicity of high-dose melphalan. Furthermore, the overall risk of cardiac event is higher in MM patients compared to age- and gender-matched individuals without MM (Kistler et al. Clin Lymph, Myel & Leuk, 2016). Pre-transplant cardiac screening may help prevent major cardiac complications such as severe heart failure, cardiac tamponade, or life-threatening cardiac arrhythmias. Supraventricular tachy arrhythmias (SVT) are a common complication which may be related to Melphalan, the transplanted stem cells or effects of para-proteinemia on the conduction system. Here, we sought to determine the incidence of SVT and its predisposing factors in this setting. Methods: We reviewed the 100-day post-ASCT clinical course of all MM patients treated from 2007 to 2017 that had undergone ASCT at University Hospitals Cleveland Medical Center. Patients also diagnosed with cardiac Light Chain Amyloidosis (AL) were excluded. Clinical data on the following characteristics were collected: patients' demographics, disease characteristics, pre-ASCT comorbidities and cardiovascular-related medication use, electrocardiography (ECG) and echocardiography (ECHO). ECG and ECHO studies was limited to one year prior to ASCT. Presence of left ventricle hypertrophy (LVH), ejection fraction (EF), impaired relaxation or abnormally low QRS voltage on pre-ASCT were recorded. QRS amplitude was measured in the V2 precordial lead as the distance from the R peak to S peak. Both atrial fibrillation and atrial flutter were classified as SVT. Continuous variables were compared using the unpaired t-test, and categorical variables were compared using the two-tailed Fisher's exact test. A multiple logistic regression model was fit to a model containing all the collected potential risk factors. Odds ratios (ORs) and confidence intervals (CIs) were estimated from the estimated betas in the model. A two-tailed p value < 0.05 was considered to be statistically significant. Results: A total of 222 patients were included in the study population. Amifostine was used before all ASCT to reduced oral mucositis. Patient median age was 60.6 (range: 36-79), 110 patients (49%) were male and the 112 female (51%). Median pre-ASCT QRS amplitude was 1.7 millivolt (mV) (range: 0.3-4.4). Median QRS duration was 90 millisecond (mS, range: 60-208). Only 166 patients received a pre-ASCT ECHO. Median time of pre-ASCT and post-ASCT ECHO were 38 days (range: 1-333 days) and 55 days (range: 4-118), respectively. Seventeen patients (8%) developed SVT during or after ASCT with a median time of 12 days post-ASCT (range: 1-97 days). Median time of the length of hospital stay between patients without SVT and with SVT was different, 15 vs. 19 days (p-value < 0.001). There was no statistically significant between average melphalan dose utilized as the conditioning regimen, infused CD34 dosage, transplant-associated weight loss, time from diagnosis or length of pre-ASCT exposure to proteasome inhibitors between two cohorts (Table-1 and 2). Also, there was no association between pre-ASCT use of anti-lipid lowering agents, beta-blockers, angiotensin-converting enzyme inhibitor or angiotensin receptor inhibitors. Out of all pre-ASCT comorbidities renal insufficiency (9% vs. 41%, p-value: 0.017) and HTN (49% vs. 76%, p-value: 0.045) were predictor, of SVT occurrence in the univariate analysis. Multivariate analysis indicated that renal insufficiency at baseline (OR: 3.6, 95% CI: 2.1-4.8, p-value < 0.001), left ventricular systolic dysfunction (LVSD, OR: 3.0, CI: 1.1-5.1, p-value: 0.003), and hypertension (OR: 2.5, CI: 1.2-4.6, p-value: 0.028), were significantly associated with the development of SVT after ASCT (Fig. 1). Conclusion: Taken together, our results suggest that SVT represents an important cause of morbidity and is associated with longer hospital stay for MM patients undergoing ASCT. The presence of abnormal renal function, LVSD, or hypertension at baseline identifies patients at greater risk of developing SVT following ASCT. Further studies are warranted to establish robust risk models that may predict SVT in MM patients prior to and during ASCT. Disclosures Caimi: ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees. Malek:Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by osteolytic bone disease and immunosuppression. Transforming growth factor-beta (TGF-β) supports MM progression through its role in stimulation of IL-6, Th17/regulatory T cell development, hematopoietic suppression, and its promotion of catabolic bone remodeling. Inhibition of the proteasome has emerged as a powerful strategy for treatment of MM. Proteasome inhibitors (PIs) have improved overall survival in patients with MM, but the majority develop drug resistance. Therefore, a novel therapeutic strategy is needed for better treatment of MM. Vactosertib (VACTOSERTIB) is an orally bioavailable small molecule inhibitor of the TGF-β type I receptor kinase (TβRI) currently in a phase I clinical trial (NCT03143985) in combination with the immunomodulatory drug, pomalidomide. In this study, we first investigate if Vactosertib is capable of diminishing myeloma progression either as a single agent or in combination with the PI, ixazomib. Methods: Expression levels of TGF-β1 and phospho-Smad2/3 was examined in human and mouse MM cells and bone marrow by Western blot and immunohistochemistry (IHC). Cellular and molecular assays were performed in human RPMI8226, ARH-77 and PIs-resistant RPMI8226 as well as murine 5T33MM cells via MTT, apoptosis, real-time PCR and Western blot. For preclinical evaluation, C57BL/KaLwRij mice bearing 5T33MM cells expressing luciferase were treated with Vactosertib, ixazomib, or the combination of Vactosertib plus ixazomib for 3 weeks, and evaluated for MM growth by bioluminescence imaging (BLI). Peripheral blood IL-6 and monoclonal protein concentration, M-spike, were measured by ELISA. Fluorescence-activated cell sorting (FACS) analyses were used for immunological evaluation, such as myeloid derived suppressor cells (MDSCs), Foxp3+ regulatory T cells (Tregs) and NK cells. Femur trabecular bone density and microarchitecture were assessed by 3D micro-CT system. Results: We first evaluated the expression of the TGF-β1 ligand and its receptor activated signaling intermediates, Smad2/3 in bone marrow of patients with MM. There is an accumulation of the type 1 isoform of the TGF-β ligand within the tumor microenvironment (TME) and of phospho-Smad2/3 in myeloma cells within the bone marrow of patients (Figure 1). In vitro studies utilizing the TbRI inhibitor Vactosertib (TEW-7197) with murine 5T33MM, human RPMI8226 (Figure 2A), U266, ARH-77 cell lines, and tumor cells from patients with MM (Figure 2B) cultured demonstrate a dose-dependent reduction in cell viability in cultures of these MM cells continuously exposed to Vactosertib. Synergistic inhibition of Vactosertib in combination with PIs was also observed in parent (Figure 2C), bortezomib- (Figure 2D), ixazomib- (Figure 2E), and carfilzomib-resistant RPMI8226 cells (Figure 2F). We also found that there is an accumulation of the TGF-β1 within the TME and phospho-Smad2/3 within the MM at advanced stages in the bone marrow of C57BL/KaLwRij mice harboring the 5T33MM myeloma cells. Oral administration of Vactosertib as a single agent inhibited MM progression as measured by mortality (Figure 3A), peripheral blood monoclonal protein concentration and BLI (Figure 3B). While the expansion of CD11b+Gr-1+ MDSCs (Fig. 3C) and the population of Tregs (Fig. 3D) in the spleen were reduced, NK cells were increased by Vactosertib alone (Fig. 3E). Combination therapy of Vactosertib plus ixazomib exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by a decrease in mortality (Figure 4A) and significant reduction in both the BLI and M-spike before and after treatment (Figure 4B, C). Furthermore, Vactosertib single therapy enhanced the bone thickness and reduced trabecular separation in comparison to control mice. Conclusions: Taken together, our data demonstrate that Vactosertib (a small molecule inhibitor of the TGF-β type I receptor kinase) suppresses viability of human MM cells, and is associated with a potent anti-myeloma effect, suppressing bone resorption and modulating the MM TME in a immunocompetent preclinical model of MM. These data provide a rationale for clinical evaluation of the combination therapy of Vactosertib and ixazomib as a potential therapeutic strategy to improve outcomes in patients with MM. Disclosures Kim: MedPacto: Other: Founder/CEO. Malek:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau.

// Ehsan Malek 1, 2 , James J. Driscoll 2, 3, 4 1 Case Western Reserve University School of Medicine, Cleveland, OH, USA 2 Division of Hematology and Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA 3 The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH, USA 4 University of Cincinnati Cancer Institute, Cincinnati, OH, USA Correspondence to: James J. Driscoll, email: driscojs@uc.edu Keywords: phosphatidylinositol 3-kinase, p110-delta, multiple myeloma, proteasome, bortezomib resistance Received: February 25, 2016 Accepted: May 04, 2016 Published: May 24, 2016 ABSTRACT Multiple myeloma (MM) remains an incurable plasma cell malignancy and drug resistance persists as the major cause of treatment failure leading to fatal outcomes. The phosphatidyl-inositol-3-kinase (PI3K) pathway is constitutively hyperactivated in MM to promote disease progression and drug resistance. While inhibiting PI3K induces apoptosis in MM and is predicted to increase tumor susceptibility to anticancer therapy, early-generation pan-PI3K inhibitors display poor clinical efficacy as well as intolerable side effects. Here, we found that PI3K activity is significantly upregulated in MM cell lines and patient tumor cells resistant to bortezomib and that the majority of PI3K activity in MM cells is dependent upon the p110-δ isoform. Genetic or pharmacologic inhibition of p110-δ substantially reduced myeloma viability and enhanced cellular sensitivity to bortezomib. Chemical library screens then identified a novel compound, DT97, that potently inhibited p110-δ kinase activity and induced apoptosis in MM cells. DT97 was evaluated in the NCI-60 panel of human cancer cell types and anticancer activity was greatest against MM, leukemia and lymphoma cells. Co-treatment with DT97 and bortezomib synergistically induced apoptosis in MM patient cells and overcame bortezomib-resistance. Although bone marrow stromal cells (BMSCs) promote MM growth, the pro-survival effects of BMSCs were significantly reduced by DT97 treatment. Co-treatment with bortezomib and DT97 reduced the growth of myeloma xenotransplants in murine models and prolonged host survival. Taken together, the results provide the basis for further clinical evaluation of p110-δ inhibitors, as monotherapy or in synergistic combinations, for the benefit of MM patients.

Although the therapeutic benefit of proteasome inhibition in multiple myeloma remains unchallenged, drug resistance inevitably emerges through mechanisms that remain elusive. Bortezomib provokes unwanted protein accumulation and the endoplasmic reticulum stress to activate the unfolded protein response (UPR) and autophagy as compensatory mechanisms that restore protein homeostasis. High-throughput screens to detect pharmacologics that modulated autophagy to enhance the anti-myeloma effect of bortezomib revealed metformin, a widely used antidiabetic agent with proven efficacy and limited adverse effects. Metformin co-treatment with bortezomib suppressed induction of the critical UPR effector glucose-regulated protein 78 (GRP78) to impair autophagosome formation and enhance apoptosis. Gene expression profiling of newly diagnosed myeloma patient tumors further correlated the hyperexpression of GRP78-encoding HSPA5 with reduced clinical response to bortezomib. The effect of bortezomib was enhanced with metformin co-treatment using myeloma patient tumor cells and the chemoresistant, stem cell-like side population that may contribute to disease recurrence. The relevance of the findings was confirmed in vivo as shown by metformin co-treatment with bortezomib that delayed the growth of myeloma xenotransplants. Taken together, our results suggest that metformin suppresses GRP78, a key driver of bortezomib-induced autophagy, and support the pharmacologic repositioning of metformin to enhance the anti-myeloma benefit of bortezomib.

Autophagy (self-eating) and apoptosis (self-killing) are evolutionarily conserved, catabolic pathways that are essential to maintain cellular and organismal homeostasis. The functional and molecular relationship between autophagy and apoptosis is highly complex since, under certain cellular circumstances, autophagy constitutes a stress adaptation to prevent or suppress apoptosis and hence cell death, whereas in other settings, autophagy constitutes an alternative pathway to promote cell death. Autophagy and apoptosis are both triggered by common upstream signals to result in a synergistic effect that stimulates these processes, or in other instances the cell switches between the two responses in a mutually exclusive manner. Therefore, the precise events and molecular effectors that regulate the complex interplay between autophagy and apoptosis as determinants of cell fate under physiologic and pathologic conditions remain poorly understood. Autophagy and apoptosis have now been shown to be connected through several nodes that display rapid, extensive cross-talk that enables their coordinated regulation. Autophagy fulfills an essential role as it facilitates the degradation of potentially oncogenic molecules, preventing the development of cancers, while apoptosis prevents the survival of cancer cells. However, mutation, gene inactivation, and other abnormalities can lead to defective autophagy and/or apoptosis and ultimately the initiation and progression of cancers. Autophagy appears to have a dual role in cancer, as it has now been shown to facilitate the survival of tumor cells in stress conditions such as hypoxic or low-nutrition environments. A more thorough and comprehensive understanding of autophagy and apoptosis is necessary to facilitate the identification of novel drug targets and to develop more effective cancer therapeutics that improve patient survival.

Significance of the absolute lymphocyte/monocyte ratio as a prognostic immune biomarker in newly diagnosed multiple myeloma

Malignant gliomas are highly aggressive primary brain tumors with few treatment options and invariably fatal outcomes. The receptor tyrosine kinase epidermal growth factor receptor (EGFR) is hyperactivated through gene amplification or mutation in many cancers. EGFR activity is associated with increased cell proliferation, disease progression and therapeutic resistance. Intratumoral heterogeneity of EGFR arises as a signature pathogenetic events in the highly aggressive and invariably fatal brain tumor glioblastoma multiforme (GBM). Functional blockade of the EGFR kinase activity alone does not achieve maximal therapeutic benefit to support the role for EGFR kinase-independent functions. We performed a high-throughput, pharmacologic screening to identify EGFR-targeted therapeutics in glioma cells engineered to overexpress either a high or low copy number of EGFR wildtype (WT), a constitutively active mutant EGFR-vIII or an EGFR kinase-dead (KD) mutant. We discovered that the heat shock protein (HSP)90 inhibitor ganetespib selectively and potently killed glioma cells that overexpressed either the WT, vIII or KD forms of EGFR (IC50 ∼20-40nM). Real-time measurements demonstrated that ganetespib reduced proliferation, induced caspase-dependent apoptosis and inhibited glioma migration. Overexpression of the EGFR-WT, vIII or KD forms significantly increased intracellular ATP levels and WT, vIII and KI EGFR all translocalized to the mitochondria. Ganetespib addition rapidly reduced ATP in cells that overexpressed either kinase-active or kinase-inactive forms. Ganetespib reduced the proliferation of human patient GBM patient-derived tumor stem cells and effectively impaired the growth of subcutaneously placed glioma implants in nude mouse models. Ganetespib impairs the EGFR kinase-(in)dependent activities with the potential benefit to overcome intratumoral heterogeneity benefit and improve GBM patient outcome.

Small molecules that modulate cancer stem cell (CSC) fate and function offer therapeutic potential to improve patient outcome. Malignant gliomas are highly aggressive primary brain tumors with few treatment options and an invariably fatal outcome. High-throughput screening and structure-guided design identified a novel, dual phosphatidylinositol-3-kinase (PI3K)/mammaliam target of rapamycin (mTOR) inhibitor that potently induced apoptosis in glioma cells and glioma patient-derived CSCs. The compound, designated DT61, inhibited PI3K, mTORC1 and mTORC2 kinase activities at nanomolar concentrations in a dose-dependent manner. DT61 dramatically reduced the level of the PI3-K α and β isoforms and led to a reduction in the site-specific phosphorylation and activity of the pro-survival serine-threonine kinase Akt/Protein Kinase B (PKB). DT61 inhibition of PI3K activity prevented the inactivation of glycogen synthase kinase (GSK-3beta) to enhance site-specific phosphorylation and proteasomal degradation of the c-myc oncoprotein that drives gliomagenesis. The apoptotic effect of DT61 on CSCs was synergistically enhanced when combined with radiation treatment, a standard-of-care modality for glioma treatment. Taken together, the results demonstrate that DT61 is a novel, highly effective dual PI3K/mTOR inhibitor that is cytotoxic to brain CSC under normoxia and hypoxia with potential benefit for glioma patients.

The ubiquitin-proteasome system (UPS) is a highly complex protein network that maintains proteostasis and cell viability through the targeted and timely turnover of selected substrates. The proteasome serves as the catalytic core of the UPS to precisely recognize and efficiently execute the rapid ATP-dependent removal of ubiquitinated proteins. Small-molecule pharmacologic inhibitors exploit the pivotal role of the proteasome in cellular metabolism as a molecular vulnerability in cancer cells to promote the selective cytotoxicity of tumor cells. Proteasome inhibitors (PIs) have yielded durable clinically responses that dramatically improve the survival of patients diagnosed with the invariably fatal hematologic malignancy multiple myeloma (MM). Success of the PI bortezomib in the treatment of the hematologic malignancy MM has emerged as the standard of care and catapulted the UPS into a position of prominence as a model system in cancer biology and drug development. However, advancement of PIs to improve the treatment of patients with solid tumors has been far more challenging and less successful. Clinical assessment of second-generation PIs progresses as well as pharmacologics to intervene at other points within the UPS is being explored for both hematologic and solid tumors. Agents to target non-proteolytic activities associated with the proteasome are emerging as are agents to inhibit Ub-binding proteins. New approaches to unravel the UPS should advance its utilization as a drug development platform in mechanism-based anticancer strategies that include PIs as monotherapy or in synergistic combinations that improve the outcome of patients with solid tumors.

Histatin 5 (Hst5) is a 24-amino acid (aa) member of the Hst family that is found in human salivary secretions and exhibits candidacidal activity. Hst5 contains a 13-aa region that alone is capable of killing fungal pathogens and is referred to as the functional domain. To investigate the role of specific aa located within the functional domain, the pRSET bacterial expression system was used to produce recombinant Hst5 (re-Hst5) and several re-variants that were generated by site-directed mutagenesis. The vector pRSETC expresses genes of interest as fusion proteins attached to the carboxy end of an N-terminal His6 tag that binds to nickel (Ni2+). The re-variants were generated using the polymerase chain reaction (PCR) and had Gly substituted for either the His, Glu or Lys/Arg within the functional domain. PCR products that encoded either the wild-type or variant forms of re-Hst5 were inserted into pRSETC and produced as fusion proteins which were affinity purified from cell lysates by Ni(2+)-Sepharose chromatography. Fusion proteins were digested with CNBr and re-Hsts were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Re-Hsts were tested in bioassays to measure the ability to kill both Candida albicans (C. albicans) blastoconidia and spheroplasts which were generated by removal of the cell wall. In both assays, re-Hst5 displayed dose-dependent candidacidal activity that was nearly identical to that of native Hst5 purified from human salivary secretions. Re-Hst5 variants with either Glu or Lys/Arg substitutions demonstrated significantly lower candidacidal activity in both assays, while the variant with His mutated showed essentially no activity at physiological concentrations. These results indicate that acidic and basic aa within the functional domain contribute to candidacidal activity and that the His are essential for candidacidal activity. Additionally, since C. albicans spheroplasts were also susceptible to Hsts, the cell wall is not an essential component in the Hst mechanism of candidacidal action.

Multiple Myeloma (MM) is an incurable plasma cell cancer that is caused by several chromosomal translocations and gene deletions. Although deregulation of several signaling pathways including the Nuclear Factor-Kappa B (NF-κB) pathway has been reported in MM, the molecular requirement and the crosstalk between NF-κB and its target genes in MM cell survival has been largely unclear. Here, we report that Yin Yang1 (YY1), a target gene for NF-κB, is hyperexpressed in most MM tumor cells obtained from human patients, exhibits constitutive nuclear localization, and is essential for survival of MM cells. Mechanistically, we report a novel YY1-RelA complex formation, which is essential to transcriptionally repress a proapoptotic gene Bim. In line with this, depletion of YY1 or RelA resulted in elevated levels of Bim and apoptosis. Moreover, both YY1 and RelA are recruited to the Bim promoter and are required to repress the Bim promoter. Importantly, depletion of YY1 or RelA almost completely impaired the colony forming ability of MM progenitor cells suggesting that both RelA and YY1 are essential for the survival and growth of MM progenitor cells. Moreover, depletion of either YY1 or RelA completely inhibited MM tumor growth in xenograft models for human myeloma. Thus, a novel RelA-YY1 transcriptional repression complex is an attractive drug target in MM.

Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite. The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule. Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hstl) that lacked phosphorylated serine. Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST). The GST/re-Hstl fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hstl from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hstl was eluted as a well-defined peak. The yield of re-Hstl was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hstl. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hstl had the same apparent molecular weights, while cationic PAGE showed that re-Hstl was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hstl lacked any detectable phosphate. Re-Hstl demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite. These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite.

Introduction: Multiple myeloma (MM) is clonal plasma cell malignancy that remains incurable. Checkpoint inhibitors represent a revolutionary form of cancer therapy that empowers the immune system to defeat cancer. Programmed death-1 (PD-1) is an inhibitory receptor expressed on immune cells, particularly cytotoxic T cells, that interacts with two ligands, PD-ligand 1 (PD-L1) and PD-L2 expressed on tumor cells. PD-L1 and PD-L2 engage PD-1 on the T cell surface to negatively modulate the magnitude of T-cell-mediated responses. Such negative feedback is critically important in maintaining homeostasis of the immune response to prevent autoimmunity during infection or inflammation in normal tissue. However, in cancers, it presents a major problem in blocking cellular antitumor responses. PD-L1/PD-L2 ligation with PD-1 provides a mechanism of immune escape for tumor cells by turning off the cytotoxic T cells. Currently available monoclonal antibodies (mAbs) that disrupt the PD-1/PD-L1 interaction have exhibited remarkable responses in selected patients and tumor types. However, mAbs demonstrate many drawbacks that include a lack of tumor cell specificity, low response rates in unselected patient populations and induction of de novoautoimmune disease that excludes many patients from therapy. We hypothesize that small molecule checkpoint inhibitors can provide greater specificity, shortened half-lives to diminish autoimmune or other adverse events, increased oral bioavailability, enhanced bio-efficacy, and higher stability at ambient temperature facilitating purification during production. Here, we identified novel, small molecule PI3K inhibitors that reduce PD-L1/PD-L2 levels on MM cells and enhance the antimyeloma activity of autologous T cells. Methods: To determine the effect of proteasome inhibitors on PD-L1/PD-L2 surface expression, RPMI8226 cells were incubated with bortezomib (BTZ), carfilzomib (CFZ) or ixazomib (IXZ) (1nM) for 36 h. Cells were then incubated in PBS/10% normal goat serum followed by antibodies (1/100) for 30 min at 4ºC and then treated with Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2. RPMI8226 cells were also incubated with the small molecule PI3K inhibitor DT97 (500nM) alone or combined with BTZ, CFZ, or IXZ. Cells were similarly stained using Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2 and a BD LSRFortessa™ cell analyzer was used for multicolor flow cytometry to acquire >10,000 events/sample. To determine the effect of DT97 on T cell-mediated cytolysis, bone marrow biopsy was performed on an MM patient and CD138+ cells were isolated by Miltenyi positive selection and CD4+ T cells by MojoSort™ CD4+ Human T Cell isolation. CD138+ cells were incubated with CD4+ T cells, proteasome inhibitors, or DT97 as indicated for 16 h at 37ºC. CD138+cells were then affinity-isolated, incubated with a FITC-conjugated anti-Annexin-V antibody and quantitated using a BD LSRFortessa™ cell analyzer. Results: Treatment of MM cells with BTZ, CFZ, or IXZ significantly increased the surface expression of PD-L1 and PD-L2 (Fig. 1). In contrast, treatment with the PI3K inhibitor DT97 suppressed the expression of PD-L1 and PD-L2 on MM cells (Fig. 2). Importantly, DT97 co-treatment with either BTZ, CFZ, or IXZ suppressed the induction of PD-L1 and PD-L2 seen after treatment with proteasome inhibitors alone. Treatment with the proteasome inhibitors alone or combined with T cells did not promote significant killing of MM cells (Fig. 3). However, DT97 treatment significantly enhanced autologous T cell-mediated MM death and the effect was further enhanced by addition of proteasome inhibitors. Conclusions: The results presented here establish the proof-of-principle that small molecule PI3K inhibitors reduce PD-L1/PD-L2 levels on tumor cells and enhance the anti-myeloma effect of autologous T cells. Small molecules checkpoint inhibitors represent a safe therapeutic alternative that can avoid the problems associated with antibodies, while retaining their functionality. Taken together, our studies indicate the promise of small molecules checkpoint inhibitors and support further translational and clinical development as a transformative form of cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

The bone marrow (BM) microenvironment contains numerous cell types, including immunosuppressive tumor-associated macrophages (TAM), that promote the growth and survival of multiple myeloma (MM) cells. TAMs are recruited to the site of MM within the BM and help to create a permissive microenvironment that promotes the homing of MM cells. Prior studies have shown that the relative amount of BM-associated TAMs correlates with patient poor outcome in MM (Suyaný et al. 2013). Emerging evidence suggests that the absolute monocyte count (AMC) in peripheral blood may reflect the level of TAM recruitment at the tumor site and its prognostic significance has been shown in other lymphoid malignancies. Absolute lymphocyte count (ALC) as a surrogate marker for host immune system has prognostic significance. Early lymphocyte recovery after induction therapy or autologous stem cell transplant correlates with better survival for MM patients. The aim of the present study was to investigate the prognostic significance of the ratio of ALC, as a surrogate of host immune status, to AMC, which reflects the MM-immune microenvironment, in predicting clinical outcome among newly diagnosed MM patients. Methods: The study included 372 MM patients (pts), diagnosed 2004-2014 at the University Hospitals in Cleveland, OH and University of Cincinnati, Cleveland OH who had CBC differentials at diagnosis. The primary end-point of the study was progression free survival (PFS) calculated from the time of diagnosis. The Cox proportional hazards model was used to evaluate ALC/AMC at diagnosis as a prognostic factor for PFS as well as to assess and adjust of other know prognostic factors. Results: Median pt age was 67.3 years (range: 30-92), 53% were male and 47% female. Distributions of detailed baseline characteristics are presented in Table-1. The median follow up was 33.5 months (range: 1.16 - 122.9). Pts who were lost to follow up were censored from the survival analysis. At the time of this analysis 92 pts (24%) had died, 75 (20%) of them was due MM. The median AMC, ALC and ALC/AMC ratio at diagnosis was 412 cells/µL, 1,461 cells/µL and 3.9, respectively. ALC neither AMC was associate with different PFS or overall survival (OS). ALC/AMC as a continuous variable was identified as a predictor of PFS [Hazard ratio (HR) = 0.62, (95% CI: 0.52 - 0.75), p-value: 0.001]. To define a cut-off point, the choice of ALC/AMC ≥ 3.6 yielded the greatest differential in survival, based on the chai-squarevalue (χ2 = 94·4, P < 0·01) analyzed at different cut-off points between the 25% and 75% quartiles (2.9 - 4.3), from the log-rank test. Pts with an ALC/AMC ≥ 3.6 (n=236) experienced a superior median PFS compared to patients with an ALC/AMC < 3.6 (n=136) [Median PFS, 24 vs. 43 months respectively, P-value < 0.001] (Figure-1). Multivariate analysis including ALC, age, ISS stage and treatment type (autologous stem cell transplant vs. no transplant) showed that ALC/AMC as well as treatment type remained an independent prognostic factor of PFS after the step-wise fashion analysis. Out of 372 patients 192 patients had cytogenetics analysis at diagnosis. Univariate analysis demonstrated that low ALC, high AMC and a low ALC/AMC ratio were correlated with presence of del (17p) and t(4,14) and light chain myeloma. Multivariate analysis indicated that the ALC and AMC were not associated with any specific cytogenetic group. However, the ALC/AMC ratio independently predict 17p and t(4,14) abnormalities (p-value = .041). Low ALC, high AMC, and low ALC/AMC ratio were associated with poor prognostic factors such as high β2-microglobulin. Conclusions: In our cohort, myeloma patients with a higher ALC/AMC at diagnosis enjoyed a longer PFS (either as a continuous or dichotomized variable). Also, multivariate analysis demonstrated that patient with tumors that harbored del (17p) or t(4, 14) had low ALC/AMC ratio suggesting possible immunoparesis. ALC at diagnosis was not associated with OS, in contrast to prior studies, possibly due to the shorter follow-up duration used here and inadequate power to predict OS. Taken together, our data potentially suggest the ALC/AMC ratio as a readily available surrogate marker to assess the relative strength of host immune system to myeloma-induced immunoparesis. The ratio can serve as a biomarker to stratify patients for future immunological therapies. Also, our findings indicate that the host immune status should be carefully monitored. Disclosures No relevant conflicts of interest to declare.

The recent number of US Food and Drug Administration (FDA) drug approvals for the treatment of multiple myeloma (MM) is unprecedented.Four new therapeutic agents,panobinostat,daratumumab,elotuzumab, andixazomibwere approved in 2015 matching the record of seven new-agents and 16 regulatory approvals during the past 12 years. New therapies have dramatically improved life expectancy for patients with MM. The therapeutic benefit of newer agents, combined with the incurable and relapse-remission nature of disease, has further propagated the interest of pharmaceuticaland academic investigations. Thus, a multitude of anti-myeloma compounds have been proposed, evaluated in pre-clinical studies and tested in phase I trials. A major hurdle to recruiting patients onto phase I oncology trials has the assumption that these trials offer low therapeutic benefit, which makes phase I trial recruitment a last resort(Simon et al. JCO, 2004).The status of MM phase I trials in the past 12 years has not been reviewed in detail. The aim of this study is to determine overall therapeutic benefit and risks forptsrecruited in phase I trials from 2004 to 2015 and to analyze the role of potential factors affecting outcomes. Methods: Phase I trials for MM conducted from 2004-2015 were identified from searches of MEDLINE, Cochrane Library and scientific meetings (Fig. 1). Data was extracted by two independent reviewers based upon the same algorithm and significant overlap in assignment to assess inter-observer heterogeneity. Results: Trials (n=74) were selected and included a total of 2408 enrolled pts (Fig. 1). The median number of pts/trial was 29 (range: 5-84), 56% of pts male, 44% female, median age was 67 years (range: 55-71) with increasing trend toward the end of the study period. The performance status was 2 or better in all trials. 39% of the trials evaluated a single agent (excluding corticosteroids) and the remainder evaluated combination therapies (Fig. 2A). 12 (42%) trials tested small molecules and 16 (68%) used monoclonal antibodies as single agent. 90% of the trials were conducted based on 3+3 design. The proportion of industry-sponsored trials increased progressively in the study period (Fig. 2B). The ratio of the initial dose to the Maximum Tolerated Dose (MTD) was 0.29 indicating significant portion of enrolled pts were potentially undertreated, most likely due to dominance of 3+3 design. 1107 of the 2408 pts responded to the study drugs which resulted in overall response rate (ORR) of 42% (range: 0-91). Median ORR was significantly different in trials with single agent vs. combination therapy (20% vs. 40%, respectively, p-value Conclusions: Our analysis indicates that the therapeutic benefit for patients recruited onto MM phase I trials was significantly higher than that reported for phase I trial of all cancer types (Horstmann et al. NEJM. 2005).Our results suggest an inverse correlation between the number of prior lines of therapies and the response rate that support earlier patient entry onto phase I studies to increase the therapeutic benefit.Also, our analysis shows that despite an increase in the number of compounds tested in MM phase I trials during the past 12 years, the overall toxicity from these trials has not increased. It is also possible that less patients would be undertreated by utilizing phase I designs other than 3+3 that deliver therapeutic dosage to a larger portion of enrolled patients. Disclosures No relevant conflicts of interest to declare.

The Ubiquitin+Proteasome System (UPS) represents a successful anti-cancer strategy that has translated from preclinical studies to clinical development and significantly improved the survival of Multiple Myeloma (MM) patients (1). The proteasome is high molecular weight structure that contains multiple proteolytic sites and functions as the catalytic core of the UPS to degrade Ub~conjugated proteins (2-4). Inhibition of the proteasome’s chymotryptic-like peptidase activity blocks the entire UPS and leads to selective tumor cell death. Bortezomib is the first proteasome inhibitor used in human clinical trials and treatment leads to a reduction in tumor burden in MM patients (5,6). However, cellular resistance to proteasome-based therapy generally results and reduces the efficacy. Resistance to proteasome inhibitors may result from altered expression of either proteasome or non-proteasome encoding genes. SUMOylation is a post-translational modification that attaches a Small Ubiquitin-like MOdifier to target proteins and, similar to Ub, may target proteins for proteasomal degradation. Gene expression profiling of MM patient samples indicated that bortezomib resistance may be achieved through induction of non-proteasome components. Importantly, certain effectors of the SUMOylation pathway were induced in the samples of MM patients that did not respond to bortezomib. A molecular-based, biologically-relevant supervised approach was used to identify a compendium of genes within the SUMO+UPS that were induced in MM patients and correlated with a decreased response to bortezomib. This molecular approach is relevant to a heterogeneous disease like MM where patients with similar clinical and pathologic features have vastly different genetic mutations responsible for disease and varying responses to therapies and clinical outcomes. The potential role of the SUMOylation pathway is only beginning to emerge, however, evidence indicates that SUMOylation is induced in myelomagenesis and also in bortezomib-resistant cells.

A concept that currently steers the development of cancer therapies has been that agents directed against specific proteins that facilitate tumorigenesis or maintain a malignant phenotype will have greater efficacy, less toxicity and a more sustained response relative to traditional cytotoxic chemotherapeutic agents. The clinical success of the targeted agent Imatinib mesylate as an inhibitor of the tyrosine kinase associated with the breakpoint cluster region-Abelson oncogene locus (BCR-ABL) in the treatment of Philadelphia-positive chronic myelogenous leukemia (CML) has served as a paradigm. While intellectually gratifying, the selective targeting of a single driver event by a small molecule, e.g., kinase inhibitor, to dampen a tumor-promoting pathway in the treatment of solid tumors is limited by many factors. Focus can alternatively be placed on targeting fundamental cellular processes that regulate multiple events, e.g., protein degradation, through the Ubiquitin (Ub)+Proteasome System (UPS). The UPS plays a critical role in modulating numerous cellular proteins to regulate cellular processes such as signal transduction, growth, proliferation, differentiation and apoptosis. Clinical success with the proteasome inhibitor bortezomib revolutionized treatment of B-cell lineage malignancies such as Multiple Myeloma (MM). However, many patients harbor primary resistance and do not respond to bortezomib and those that do respond inevitably develop resistance (secondary resistance). The lack of clinical efficacy of proteasome inhibitors in the treatment of solid tumors may be linked mechanistically to the resistance detected during treatment of hematologic malignancies. Potential mechanisms of resistance and means to improve the response to proteasome inhibitors in solid tumors are discussed.

Bortezomib is the first therapeutic inhibitor of the proteasome that has demonstrated a significant clinical response in patients with otherwise refractory or rapidly advancing disease. Bortezomib has received US Federal Drug Administration approval for the treatment of the hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Herein, the use of bortezomib as an upfront therapy, as an induction regimen before stem-cell transplantation and as maintenance therapy in the treatment of multiple myeloma is discussed.

The ubiquitin (Ub)+proteasome proteolytic pathway is responsible for the selective degradation of the majority of nuclear and cytosolic proteins. The proteasome is a high molecular weight multicatalytic protease that serves as the catalytic core of the complex Ub-dependent protein degradation pathway and is an exciting new target for the development of novel anticancer therapies. Inhibition of the proteasome, and consequently Ub-dependent proteolysis, with the small molecule pharmacologic agent bortezomib led to approval by the US Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) that has subsequently been extended to other hematologic malignancies. Inhibition of the proteasome results in the intracellular accumulation of many ubiquitinated proteins that control essential cellular functions such as cellular growth and apoptosis. The accumulation of high molecular weight Ub~protein conjugates eventually triggers apoptosis, with tumor cells more susceptible to proteasome inhibition than non-malignant cells. The defined mechanism of action for proteasome inhibitors has not been completely characterized, not all patients respond to proteasome inhibitor-based therapy, and inevitably patients develop resistance to proteasome inhibitors. Further investigation of the Ub+proteasome system (UPS) is needed to develop more effective inhibitors, to develop agents that overcome bortezomib resistance and to avoid adverse effects such as neuropathy. Furthermore, there are newly uncovered pathways, e.g., the SUMOylation and NEDDylation pathways, which similarly attach Ub-like proteins (ULPs) to protein substrates. The functional consequence of these modifications is only beginning to emerge, but these pathways have been linked to tumorigenesis and may similarly provide therapeutic targets. The immunoproteasome is a specialized form of the proteasome that produces peptides that are presented at the cell surface as major histocompatibility complex (MHC) class I antigens. Proteasome inhibitors decrease the presentation of antigenic peptides to reduce tumor cell recognition by cytotoxic T cells (CTLs) but unexpectedly increase tumor cell recognition by natural killer (NK) cells. Inhibitors of the UPS are validated, cytotoxic agents that may be further exploited in immunotherapy since they modulate tumor cell recognition by effectors of the immune system. Targeting the UPS, SUMOylation and NEDDylation pathways offers great promise in the treatment of hematologic and solid malignancies.

The proteasome serves as the catalytic core of the Ubiquitin (Ub) protein degradation pathway and has become an intriguing target in drug development and cancer therapy. Successful pharmacologic inhibition of the proteasome with the small molecule bortezomib led to US Food and Drug Administration (FDA) regulatory approval for the treatment of mantle cell lymphoma and multiple myeloma (MM) and has been extended to a steadily increasing number of clinical trials to assess effi cacy and safety in other hematologic malignances and solid tumors. Proteasome inhibition results in the accumulation of multi-ubiquitinated proteins, which are normally degraded through the tightly regulated Ub pathway. The Ub-Proteasome pathway is responsible for the selective degradation of many proteins that regulate the cell cycle and growth. Inhibition of the proteasome generates the accumulation of multi-ubiquitinated proteins that eventually leads to apoptosis although the exact mechanism of cell death is not completely understood. A specialized form of the proteasome, known to as the immunoproteasome, processes intracellular and viral proteins to generate peptides that are then presented at the cell surface bound as antigens (Ags) bound to the Major Histocompatibility Complex (MHC) class I molecule receptor. Importantly, inhibitors of the immunoproteasome decrease the processing and generation of MHC class I Ags and alter tumor cell recognition by the principal cellular effectors of the immune system. Hence, proteasome inhibitors may be employed as therapeutics to regulate the production of tumor specifi c Ags and for the selective removal of tumor cells through recognition by cytotoxic T lymphocytes (CTLs), natural killer (NK) cells and dendritic cells (DC). Proteasome inhibitors have been validated as effective cytotoxic agents and may have further potential as novel immunotherapeutic strategies.

Overall survival (OS) is the gold standard primary end point to evaluate the outcome of any drug, biologic, intervention, or procedure that is assessed in oncologic clinical trials. OS is universally recognized as being unambiguous, unbiased, with a defined end point of paramount clinical relevance, and positive results provide confirmatory evidence that a given treatment extends the life of a patient. Clinical trialists relentlessly attempt to devise more easily measured, cost-effective, and readily available event-driven end points as predictive surrogates of a definitive outcome, such as OS, and reduce the time with which clinical trials deliver definitive results. For some treatment modalities used in a limited number of cancer types, certain event-driven surrogates, eg, progression-free survival or time-to-progression may predict OS benefit. Biologic, cell-based, and vaccine-generated treatments are rapidly expanding the oncologist's armamentarium to combat cancers and pose a dilemma in that response may not be reflected by progression-free survival, time-to-progression, or other surrogates? As targeted therapies march forward, will each new therapy require a unique biomarker validated for every disease indication? Moreover, adjuvant treatments have demonstrated efficacy and given the current limited possibility of cure in the metastatic setting, should other end points, eg, quality-of-life, emerge as valid outcomes to demonstrate benefit. Clinical trials must continue to assess OS until biologically plausible measures are developed and emerge as valid early end point surrogates to replace the gold standard.

Alzheimer's disease, Down's syndrome, and to a far lesser extent, normal aged brains exhibit abnormal extracellular deposits of amyloid. The major component of brain amyloid is the beta-protein, a 4Kd fragment of the larger beta-protein precursor. The finding of the abnormally processed beta-protein and a protease inhibitor (alpha 1-antichymotrypsin) in the amyloid deposits prompted us to search for proteases which may generate the beta-protein from its precursor. We now report on the presence and partial purification of one such proteolytic activity from Alzheimer's brain. Normal physiologic C-terminal cleavage of the secreted form of the beta-protein precursor occurs in the middle of the beta-protein suggesting that the beta-protein accumulates due to an alternative degradation pathway. We propose here that the protease activity we describe participates in this abnormal pathway.

Mammalian cells contain two large proteolytic complexes, the 650-kDa proteasome (or multicatalytic protease) and the 1500-kDa (26 S) Ubiquitin-conjugate-degrading enzyme. Since the proteasome is also required for the ATP-dependent degradation of ubiquitinated proteins, we tested whether it may be a component of the larger complex. The proteasome normally is soluble in 38% ammonium sulfate. However, after preincubation of reticulocyte extracts with ATP, several proteasome activities appeared in the 38% ammonium sulfate pellet, including the ability to degrade hydrophobic peptides and 14C-casein. Also, following preincubation with ATP, the precipitable fraction could degrade 125I-lysozyme-ubiquitin (Ub) conjugates. The activities were not present after incubation without ATP or with a nonmetabolizable ATP analog. Nondenaturing gel electrophoresis indicated the ATP-dependent appearance of a new band which degraded proteasome substrates, and reacted with an anti-proteasome monoclonal antibody on Western blot. This new band appeared larger than the proteasome and migrated similarly to the larger Ub-conjugate-degrading complex. The formation of the larger complex required factor(s) present in the 38% ammonium sulfate pellet and either the 40-80% fraction or the purified proteasome from reticulocytes or muscle. After complex formation, hydrolysis of Ub-protein conjugates and also the non-ubiquitinated substrate, casein, was stimulated severalfold by ATP, but non-metabolizable ATP analogs had little or no effect. Thus, the proteasome corresponds to component CF-3 of Ganoth et al. (Ganoth, D., Leshinisky, E., Eytan, E., and Hershkov, A. (1989) J. Biol. Chem. 263 12412-12419) and undergoes an energy-dependent association with other factors to form the 1500-kDa, ATP-requiring proteolytic complex.

Background: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT), performed either at the time of initial diagnosis or at relapse, is considered the standard of care for younger patients ( Methods: The primary decision examined in this study is the timing of ASCT in relation to a phase I trial. We systematically reviewed the outcome of all MM phase I trials between 2004-2014 to build a comparable cohort for decision analysis. Response rate, adverse effects and mortality from single agents, as well as the combination of the experimental agents with immunomodulators (IMiDs) and proteasome inhibitors (PIs) are reported. Two strategies were tested through Markov modeling: early ASCT vs. late ASCT (Figure-1). The model was built using the commercially available DecisionMaker software. An annual discount rate of 3% was used for all clinical outcomes. Quality of life (QOL) information for post-ASCT and phase I trials were extracted from similar research papers. Results: Phase I systematic review: A total of 43 phase I clinical trials with 946 patients (530 males and 416 females) were included. The precipitants' median age was 60 years old. 21 trials tested single agents and the remainder were done using combination therapies (i.e., with an IMiD or PI). Median overall response rate (ORR) was 34% for all trials, 16% with single agents and 42% with combination therapy (Figure-2). 89% of trials were completed in less than a year. The therapeutic benefit of single vs. combination therapy phase I clinical trials does not show an increasing trend during the last decade (Figure-3). Patients who participated in combination therapy phase I trials had more grade III-IV toxicity than single agents (HR: 1.35, p-value: 0.04). There were only 4 patients (less than 1% of all participants) who experienced therapy-related death. Although, time from diagnosis was a predictor of response in univariate analysis, but it was not statistically correlated with response by multivariate analysis. Also, ORR was not affected by the number of prior therapies. Markov model: The discounted life expectancies for two strategies were calculated; fixed-time intervals of 6, 12, 18 and 24 months were chosen for analysis. The gains in life expectancy were the same in patients undergoing combination therapy phase I trials in early or late ASCT strategies (6.76 and 6.64, respectively; p-value: 0.21). Importantly, for patients undergoing single agent phase I therapy, early ASCT was associated with a higher life expectancy than was the strategy of delayed ASCT (7.96 vs. 6.86, respectively; p-value: 0.036). Conclusions: Taken together, our study indicates that phase I trials demonstrate a higher ORR than reported response rates from traditional chemotherapy phase I trials, i.e. 5%, Horstmann et al. 2005, even with single agents. We conclude that phase I participation should not be viewed as the "last resort". Our decision-making analysis shows that combination therapy phase I trials can be offered irrespective of ASCT. However, single agent phase I trials should be offered after ASCT for transplant-eligible patients. Figure 1. Markov decision model. A relapsed MM base case transitions after each one-month cycle to other health states. Patients could have remained in an alive state for any number of cycles without transitioning to another health state. The ASCT and Phase I states are transitory states. Figure 1. Markov decision model. A relapsed MM base case transitions after each one-month cycle to other health states. Patients could have remained in an alive state for any number of cycles without transitioning to another health state. The ASCT and Phase I states are transitory states. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Off Label Use: Panobinostat and Ixazomib combined in myeloma.

Background: Immunochemotherapy has improved the clinical outcome of patients diagnosed with diffuse large B-cell lymphoma (DLBCL), but only 60% of all DLBCL patients are potentially cured. A response-adaptive imaging strategy that accurately determines the initial response to therapy could improve clinical outcomes. Although there has been an increasing trend to perform interim PET/CT to monitor response, the optimal interpretation method for interim PET analyses remains uncertain. Studies using maximum standard uptake value (SUVmax), have not defined a uniformly applicable SUVmax reduction cutoff that accurately predicts clinical outcome. Here, we hypothesized that a method that maximized the detection of all metabolically active regions within the tumor mass, defined as the metabolic tumor volume (MTV), could serve as a better predictor of clinical outcome than SUVmax measurement. Methods: All patients with DLBCL that were treated from Dec 2006 to Dec 2014 at the University of Cincinnati were studied, retrospectively. Interim PET analysis was performed after 2-4 cycles of chemotherapy. SUVmax and MTV on the initial and interim PET CT for each patient was determined. To evaluate the contribution of metabolic activity within the tumor periphery in assessing clinical outcomes, MTV was measured by two separate methods: fixed-threshold and gradient-segmentation using MIMSoftware, OH, USA. The primary end point of the study was progression free survival (PFS). To identify an optimal threshold cutoff that could predict PFS more accurately, the receiver operating characteristic (ROC) curve analysis was used. Results: A total of 197 patients with pathology confirmed diagnosis of DLBCL were recognized. Of the 197 patients, 167 underwent interim PET analysis. All patients fasted >6 h before intravenous injection of 18F-glucose, had glucose levels >90 and 48 h before imaging. The median follow-up period for patients in the study was 37 months. R-CHOP (Rituximab, Cyclophosphamide, doxorubicin/Hydroxydaunomycin, vincristine/Oncovin and Prednisone) and R-DA-EPOCH (Dose-Adjusted Etoposide, Prednisone, Oncovin, Cyclophosphamide and Hydroxydaunorubicin) were the first line of therapy in 74% and 26% of patients, respectively. On interim PET/CT, 69% of patients achieved complete response with the remaining patients showing partial response based on visual assessment. Dichotomous visual interpretation of interim PET did not correlate with PFS (log-rank P= 0.37). Compared with the threshold-based method, the gradient-based method resulted in a statistically significant greater MTV in pretreatment, as well as interim PET images. However, no significant difference was noted between the reduction in MTV determined by the threshold-based (ΔMTVT) or gradient-based (ΔMTVG) methods (median 34% vs 36%, P =0.29). Thresholds of ΔSUVmax and ΔMTV by ROC curve were 72% and 52%, respectively. ΔMTV predicted PFS better than ΔSUVmax as the AUC for ΔMTV was significantly larger compared with that for ΔSUVmax (Figure 1). All patients who achieved a SUVmax reduction greater than the cutoff value determined by the ROC analysis (ΔSUVmax>72%) were then stratified into two groups based on a ΔMTV cutoff value > or 72% on interim PET/CT imaging, 77 (67%) had a ΔMTV >52%. Importantly, patients who achieved a ΔMTV >52% had a statistically significantly greater PFS compared with patients who achieved a ΔMTV 72% on interim PET and those who demonstrated a ΔMTV >52% exhibited greater PFS. Conclusion: Our study highlights the importance of MTV assessment for patients who achieved background SUVmax on the interim PET to better predict the clinical outcome of DLBCL patients. The MTV measurement with the gradient-based method renders a larger volume of tumor than the threshold-based method; however, these two methods report a similar percentage reduction and are interchangeable in terms of interim PET interpretation. Metabolic activity of the peripheral area of the tumor should be incorporated into response-adaptive strategies and prospective trials that evaluate the response to current and novel therapeutic regimens to treat DLBCL patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance. Disclosures No relevant conflicts of interest to declare.

Multiple Myeloma (MM) is a complex disease that frequently leads to fatal outcome and accurate risk classification to optimize the choice of therapy may have impact on clinical outcomes. MM stratification based upon cytogenetic abnormalities and protein-coding gene expression does not adequately correlate with the depth or durability of therapeutic response. Therefore, the new class of molecular effectors, non-coding RNAs (ncRNAs), may enhance the basic understanding of myelomagenesis, drug resistance and provide better stratification of myeloma subtypes. NcRNAs include long ncRNAs (lncRNAs) and microRNAs (miRNAs) that may act as positive or negative regulators of gene expression to control cell proliferation, apoptosis and drug resistance. We hypothesized that lncRNAs commonly deregulated in the 3 resistant cell lines would have significant overlap to the deregulated lncRNA in MM patients. To investigate the role of lncRNAs in resistance to proteasome inhibitors (PIs), we compared global lncRNA profiling in drug-naïve cells to cells with acquired resistance to the PIs bortezomib, carfilzomib and ixazomib. RPMI 8226 cells resistant to each of the three PIs were generated through successive exposure to bortezomib, carfilzomib or ixazomib. Total RNA was isolated and genome-wide ncRNA expression profiling was performed using Affymetrix 3.0 microarray chips LncRNA expression profiles from drug-resistant cells were compared to that of drug-naïve cells treated with vehicle alone. MM patients’ bone marrow aspirates were obtained after UCCOM IRB-approval. Bioinformatic analysis identified a panel of 18 lncRNAs that were significantly (>100-fold) deregulated in all three drug-resistant cell lines relative to drug-naïve cells. Strikingly, the majority of the deregulated lncRNAs exhibited a similar expression pattern in all 3 PI-resistant cell lines (Figure 2a). RPMI 8226 cells carry a t(14,16) and none of the deregulated lncRNAs detected localized to chromosomes 14 or 16, suggestive of a cytogenetic-independent mechanism of drug resistance. We also identified lncRNAs deregulated in MM patient samples relative to plasma cells from healthy age-matched individuals. We found a significant overlap (>90%) between deregulated lncRNAs in PI-resistant cells and MM cells. The lncRNA COL4A-2A was upregulated >5,000-fold in resistant cells and displayed extensive sequence complementarity to miRNA-29 that was downregulated in resistant cells. Taken together, we identified a curated panel of deregulated lncRNAs in common within myeloma cells generated with acquired resistant to three different clinically-relevant proteasome inhibitors and MM patients. Further investigation is warranted to shed light on the role of these lncRNAs in the development of MM, to identify their targets and to define their role in drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Rebekah Karns, Anil G. Jegga, Sajjeev Jagannathan, Nikhil Vad, Mohamed A.Y Abdel Malek, James J. Driscoll. Long non-coding RNAs deregulated in Multiple Myeloma impact therapeutic response to proteasome inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2015-153

Multiple myeloma (MM) patients invariably relapse through mechanisms that remain elusive. Disease recurrence after treatment suggests that rare, tumor-initiating subpopulations with chemoresistant phenotypes persist or emerge to propagate and mediate tumor regrowth. Stem cell-like side population (SP) cells were isolated from myeloma cell lines and patient samples and exhibited resistance to proteasome inhibitors relative to the main population (MP). SP cell-based high-throughput screening detected pharmacologics that enhanced the anti-proliferative effect of bortezomib and revealed the anti-diabetic agent metformin. Bortezomib treatment increased levels of the molecular chaperone glucose-regulated protein GRP78 but metformin co-treatment suppressed bortezomib-induced GRP78 upregulation. Bortezomib treatment also promoted GRP78 co-localization with the signal transducer phosphatidylinositol-3-kinase (PI3K) that activates cell growth and survival pathways. SP cells displayed greater levels of the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and enhanced PI3K-mediated phosphorylation of protein kinase B/AKT compared to MP cells. Metformin treatment or knockdown of GRP78-encoding HSPA5 enhanced sensitivity to bortezomib preferentially in SP cells. Tumor growth was significantly delayed and overall survival prolonged after metformin co-treatment with bortezomib in mice injected with parental or SP cells. Taken together, the results support the pharmacologic repositioning of metformin to inhibit GRP78 induction in therapy-resistant cells and enhance the anti-myeloma benefit of bortezomib. Note: This abstract was not presented at the meeting. Citation Format: James J. Driscoll, Sajjeev Jagannathan, Mohamed AY Abdel Malek, Nikhil Vad, Ehsan Malek. Metformin suppresses GRP78-dependent PI3-Kinase activity in clonogenic side population to enhance the anti-myeloma benefit of bortezomib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4242. doi:10.1158/1538-7445.AM2015-4242

The molecular events that dictate the initiation, progression and therapeutic response in multiple myeloma (MM) remain incompletely understood. The ubiquitin (Ub) ligase Skp1-Cullin-1-Skp2 (SCFSkp2) complex promotes proliferation by inducing proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1. Skp2 overexpression and reduced p27 levels are frequent events in human cancers and are associated with poor prognosis. Methods: Prospective pharmacogenomics was performed using next generation exome sequencing gene expression profiles (GEPs) compiled from the APEX/SUMMIT phase III clinical trial deposited in dataset GSE9782. 255 relapsed MM patients were randomized to bortezomib (BTZ) vs. dexamethasone (DEX) alone and progression-free (PFS) and overall survival (OS) then correlated with the pre-treatment GEPs. RPMI8226 cells were exposed for 8 months to successively escalated doses of the proteasome inhibitors BTZ, carfilzomib or ixazomib to generate chemoresistant cell lines. Side population (SP) cells were isolated from MMCLs and patients’ samples by flow cytometry sorting based upon Hoechst 33342 staining. Results: Pharmacogenomic correlation of GEPs with clinical response revealed hyperexpression of CUL1 and SKP2 in tumors from MM patients that did not respond to BTZ. CUL1 and SKP2 hyperexpression was correlated with significantly reduced PFS and OS after treatment with BTZ but not with DEX. Cullin-1 and Skp2 were elevated in cells generated with acquired PI-resistance. CUL1 or SKP2 genetic ablation significantly enhanced the sensitivity of chemoresistant cells to PIs. A high-throughput assay was then employed using GFP-tagged p27 to screen large databases and chemical libraries in order to identify novel lead compounds that inhibited the Skp2-dependent ubiquitination of p27. Treatment with DT204, a novel lead compound identified here, reduced p27 ubiquitination, its accumulation in myeloma cells and impaired Skp2 association with Cullin-1 and Cks1, a critical rate-limiting effector of Skp2 activity. The anti-myeloma effect of DT204 was diminished using a phosphorylation-defective p27 mutant. Co-treatment with DT204 enhanced the anti-myeloma effect of BTZ against MM cell lines, patient tumor cells, stem cells and the tumor-initiating clonogenic side population (SP). The in-vivo efficacy and survival benefit of DT204 as monotherapy or in combination with BTZ will be reported. Conclusion: Taken together, the results demonstrate that pharmacologics to selectively disrupt the SCFSkp2 complex-p27 axis within the UPS holds promise to overcome chemoresistance with therapeutic benefit for myeloma patients. Skp2, Cullin1 can be utilized as biomarkers to select MM patients who will benefit from early experimental therapeutics of Skp2-inhibitors. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Mohamed Abdel Malek, James J. Driscoll. Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-245. doi:10.1158/1538-7445.AM2015-LB-245

Metabolic tumor volume on interim PET is a better predictor of outcome in diffuse large B-cell lymphoma than semiquantitative methods

8585 Background: Despite the therapeutic benefit of novel agents, majority of multiple myeloma (MM) patients inevitably relapse leading to poor clinical outcomes. During the past decade, there has ...

e13510 Background: While anti-myeloma therapies are effective in eradicating the bulk of tumor cells, intrinsically drug resistant, clonogenic tumor-initiating subpopulations contribute to disease ...

Proteasomes are highly conserved macromolecular structures which function as endopeptidases. They are found in the cytoplasm and nucleus of eukaryotic tissues and consist of at least 14 non-identical subunits with molecular masses ranging from approximately 20 to 32K. Proteasomes are essential in the selective degradation of ubiquitinated and certain non-ubiquitinated proteins, acting as the proteolytic core of an energy-dependent 26S (1,500K) proteolytic complex. Two proteasome subunits, LMP2 and LMP7 (refs 4-7), are encoded within the major histocompatibility complex (MHC), implicating proteasomes in antigen processing. Here we determine the function of these two MHC-linked subunits by comparing the proteolytic activities of purified proteasomes containing (LMP+) or lacking (LMP-) these components. We find that proteasomes of both types have endopeptidase activity against substrates bearing hydrophobic, basic or acidic residues immediately preceding the cleavage site (the P1 position) and at sites following asparagine, glycine and proline residues. The activity of LMP+ proteasomes is much higher than that of LMP- proteasomes against substrates with hydrophobic, basic or asparagine residues at P1, whereas their activities are comparable when acidic and glycine residues are present at P1. The MHC-linked LMP2 and LMP7 subunits therefore function to amplify specific endopeptidase activities of the proteasome.

Background: Multiple myeloma (MM) is a malignancy of post-germinal center B-cells whose pathogenesis remains poorly understood and that remains invariably fatal. Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches and contributes to drug resistance. Despite the therapeutic benefit of the proteasome inhibitor bortezomib, drug resistance still accounts for the majority of tumor relapses and cancer-related deaths in MM patients. A precise understanding of the molecular mechanism(s) of resistance to bortezomib in myeloma is lacking. The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin (Ub)+proteasome system (UPS) and also disrupt the aggresome-autophagy pathway for anti-myeloma benefit. Functional blockade of the UPS using targeted therapeutics has translated into clinical success and transformed the management of MM. However, drug resistance emerges through induction of the aggresome-autophagy pathway as a compensatory protein clearance mechanism leading to treatment failure, disease relapse and ultimately fatal outcome. Methods: Myeloma cells were separately exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was then performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naïve cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly deregulated in the drug-resistant cells. To determine the effect of individual miRNAs on myeloma cells, synthetic miRNA replacements or antagomirs were transfected into myeloma cells. The effect of the single miRNAs on the growth and drug resistance was then quantified using cell viability assays. Individual miRNA replacements or antagomirs were also transfected into human embryonic kidney (HEK) cells that expressed a short-lived green fluorescent protein that is degraded by the proteasome. The effect of individual miRNAs on the UPS was then quantitated in myeloma or HEK cells by confocal microscopy and fluorescent imaging. Results: MiRNA-29b was significantly downregulated in the myeloma cells generated with acquired resistance to bortezomib, carfilzomib and ixazomib relative to the parental cells. MiRNA-29b was also downregulated in MM patient bone marrow-derived tumor cells relative to plasma cells obtained from healthy individuals. MiRNA-29b targets PSME4which encodes the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29b replacements reduced the viability of myeloma cells and patient tumor cells and synergistically enhanced the cytotoxic effect of proteasome inhibitors. The miRNA-29b replacement also reduced the growth of myeloma xenografts in mouse models in vivo. MiRNA-29b replacements decreased expression of PSME4and the protein product PA200, a proteasome activator. The reduction of PA200 levels inhibited the proteasome’s chymotrypsin-like peptidase activity and the turnover of ornithine decarboxylase, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble Ub-conjugated proteins, termed aggresomes, accumulate and are degraded by the autophagy pathway. Importantly, in contrast to bortezomib, miRNA-29b replacement inhibited proteasome activity but did not induce the formation of perinuclear aggresomes or autophagosomes. Rather, immunofluorescence studies indicated that miRNA-29b replacement promoted the accumulation of small, ubiquitinated protein aggregates that were dispersed throughout the cytoplasm but were not transported by microtubules and were not coalesced into mature aggresomes. Ub-conjugated proteins were also not detected in autophagosomes. Conclusions: MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and to disrupt the efficient delivery of Ub-conjugate-containing aggresomes to the autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma benefit of proteasome inhibitors. Disclosures No relevant conflicts of interest to declare.

Background: Functional blockade of the ubiquitin (Ub)+proteasome system (UPS) using the small molecule bortezomib represents a remarkable bench-to-bedside success that has improved the outcome of patients diagnosed with multiple myeloma (MM). However, certain patients do not respond to bortezomib and those that do inevitably develop resistance through mechanisms that remain elusive and despite all currently available therapies, MM remains invariably fatal. While bortezomib disrupts protein homeostasis leading to tumor cell death but also activates pro-survival aggresome and autophagosome formation as compensatory protein clearance mechanisms to promote the emergence of drug resistance, tumor survival and disease relapse. While the therapeutic benefit of targeting the proteasome is unchallenged, more precise modalities that do not induce drug resistance are urgently needed. These studies were performed to identify pharmacologics that inhibited the autophagy pathway and to uncover their mechanisms of action as a means to synergistically enhance the cytotoxic effect of bortezomib for myeloma treatment. Methods: High-throughput screening (HTS) was performed to identify FDA-approved pharmacologic agents and bioactive molecules that prevented bortezomib-mediated induction of autophagosomes. Confocal microscopy combined with novel, dye-based fluorescent methods were used to visualize and quantitate aggresomes and autophagosomes. The effect of pharmacologics on aggresome and autophagosome formation was then determined. The effects of metformin, alone or combined with bortezomib were evaluated using MM cell lines, myeloma patient bone marrow-derived tumor cells and myeloma cells xenotransplanted into mouse models. Results: Bortezomib inhibits the UPS to promote the unwanted intracellular accumulation of ubiquitinated proteins that form aggregates dispersed throughout the cytoplasm. These aggregates are then coalesced into perinuclear aggresomes and subsequently disposed through autophagosome formation and the lysosomal pathway. Bortezomib treatment of myeloma cells increased levels of the endoplasmic reticulum (ER) molecular chaperone Glucose-Regulated Protein 78 (GRP78), a critical regulator of the unfolded protein response (UPR) and autophagosome formation. HTS revealed that the anti-diabetic biguanide metformin inhibited bortezomib-mediated autophagosome formation. Co-treatment with metformin and bortezomib increased the level of proteotoxic aggresomes but suppressed GRP78 and autophagosome formation. Metformin synergistically enhanced the anti-proliferative effect of bortezomib in myeloma cells and bone marrow-derived patient PCs. Co-treatment with metformin and proteasome inhibitors also overcame drug-resistance. shRNA-mediated knockdown of HSPA5, which encodes GRP78, similarly enhanced bortezomib-induced aggresome formation and blocked autophagosome formation. A GRP78 mutant that lacked the carboxy-terminal KDEL ER-retention motif also led to bortezomib-induced aggresomes without autophagosomes. Metformin and bortezomib together reduced myeloma xenotransplant growth in mice more effectively than either agent administered as single-agent. Conclusions: The ER chaperone GRP78 facilitates aggresome clearance by promoting autophagosome formation. Although bortezomib alone promotes GRP78 induction and aggresome formation, metformin suppresses GRP78 to uncouple the cytotoxic aggresomes from the autophagy pathway and to enhance the anti-myeloma effect of bortezomib. The results support the pharmacologic repositioning of metformin in combination with bortezomib as an effective anti-myeloma strategy. The molecular chaperone GRP78 holds promise as a target for cancer treatment as well as for neurodegenerative diseases in which intracellular protein aggregates are central to disease pathogenesis Disclosures No relevant conflicts of interest to declare.