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Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor (NR) family of transcription factors with important regulatory roles in cellular growth, differentiation, and apoptosis. Using proteomic analysis, we showed expression of PPARγ protein in a series of 260 newly diagnosed primary acute myelogenous leukemia (AML) samples. Forced expression of PPARγ enhanced the sensitivity of myeloid leukemic cells to apoptosis induced by PPARγ agonists 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-12,14-15DPGJ2, through preferential cleavage of caspase-8. No effects on cell cycle distribution or differentiation were noted, despite prominent induction of p21 in PPARγ-transfected cells. In turn, antagonizing PPARγ function by small interfering RNA or pharmacologic PPARγ inhibitor significantly diminished apoptosis induction by CDDO. Overexpression of coactivator protein DRIP205 resulted in enhanced differentiation induction by CDDO in AML cells through PPARγ activation. Studies with DRIP205 deletion constructs showed that the NR boxes of DRIP205 are not required for this coactivation. In a phase I clinical trial of CDDO (RTA-401) in leukemia, CDDO induced an increase in PPARγ mRNA expression in six of nine patient samples; of those, induction of differentiation was documented in four patients and that of p21 in three patients, all expressing DRIP205 protein. In summary, these findings suggest that cellular levels of PPARγ regulate induction of apoptosis via caspase-8 activation, whereas the coactivator DRIP205 is a determinant of induction of differentiation, in response to PPARγ agonists in leukemic cells. Cancer Res; 70(12); 4949–60. ©2010 AACR.

Supplementary Figures 1-4 from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Supplementary Figure Legends 1-6, Methods, Table 2 legend from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Supplementary Figure 5 from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Supplementary Table 1 from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Eukaryotic cells have two separate genomes; nuclear chromosomal DNA and circular mitochondrial DNA. Mitochondrial DNA lacks introns, and encodes 2 rRNAs, 22 t-RNAs and 13 of the 90 proteins that constitute the mitochondrial respiratory chain. To maintain homeostasis, mitochondrial RNA degradation machinery regulates RNA turnover. ATP-dependent helicase, SUV3 (gene SUPV3L1), and exonuclease PNPase (gene PNPT1) function as a complex to degrade mitochondrial dsRNA. By immunoblotting, PNPase and SUV3 proteins were increased in 7/7 AML patient samples and 13/13 of AML cell lines, compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall and event free survival. Genetic knockdown or knockout using shRNA or sgRNA against PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML 8227 cells. Furthermore, SUPV3L1 & PNPT1 ranked in the top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (depmap.org). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells and significantly reduced engraftment of TEX cells into the marrow of immune deficient mice, demonstrating the functional importance on leukemia initiating cells in vivo. SUPV3L1 knockdown in primary AML cells reduced engraftment in marrow of immune deficient mice. Bioinformatics analysis to detect processes associated with PNPT1 and SUPV3L1, we identified associations with Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. Consistent with this, we observed knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) suggesting an interferon response. As PNPT1 and SUPV3L1 degrade mitochondrial dsRNA, we measured levels of dsRNA after knockdown of these genes. Knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic/mitochondrial dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased dsRNA in 143B cells, but not Rho (0) 143B cells that lack mitochondrial DNA. Upregulation of inflammatory genes leads to viral mimicry and can increase sensitivity to immune mediated killing. We observed enhanced sensitivity to Double Negative T (DNT) cells mediated killing in PNPT1 and SUPV3L1 knockdown OCI-AML2 cells compared to control cells. In summary, RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML, and are essential for the survival of AML cells and AML stem/progenitors. These enzymes regulate levels of mitochondrial dsRNA, and their inhibition leads to increased cytoplasmic dsRNA triggering a viral mimicry response and enhanced sensitivity to immune-mediated killing. Citation Format: Geethu Emily Thomas, Kazem Nouri, Jong Bok Lee, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiao Ming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Li Zhang, Vito Spadavecchio, Aaron Schimmer. Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4833.

The impact of adverse risk genetic profiles on outcomes in acute myeloid leukemia (AML) patients following allogeneic hematopoietic stem cell transplantation (HCT) has not been fully elucidated. Accordingly, we have profiled somatic mutations at diagnosis using next-generation sequencing (NGS) in 178 AML patients who received allogeneic HCT. NGS revealed 598 somatic mutations in 165/178 patients (92.7%). Frequently mutated genes include DNMT3A, TET2, NPM1, RUNX1, IDH2, and FLT3. Commonly detected cytogenetic profiles include normal karyotype, trisomy 8, monosomal karyotype (MK), deletion 5, complex karyotype (CK), and monosomy 7. In univariate analyses, TP53 mutation, MK, CK, and monosomy 7 were associated with decreased overall survival (OS), relapse-free survival (RFS), and a higher relapse incidence (RI). We defined adverse molecular-genetic profile as harboring at least one of the molecular/genetic abnormalities of TP53 mutation, MK, CK, monosomy 7, and deletion 5. The patients harboring adverse molecular-genetic profile (n = 30) showed a lower 2-year OS (24.9% vs. 57.9%; p = 0.003), RFS (23.7% vs. 57.9%; p = 0.002), and higher RI (47.2% and 17.2%; p = 0.001) after HCT when compared to patients without those lesions. Multivariate analysis confirmed adverse molecular-genetic profile as an independent prognostic factor, associated with decreased OS (HR 2.19), RFS (HR 2.23), and higher RI (HR 2.94).

Efficient and accurate replication and repair of mitochondrial DNA is essential for cellular viability, yet only a minimal complement of mitochondrial proteins with relevant activities have been identified. Here, we describe an approach to screen for new pathways involved in the maintenance of mitochondrial DNA (mtDNA) that leverages the activities of DNA-damaging probes exhibiting specific subcellular localization. By conducting a siRNA screen of known nuclear DNA maintenance factors, and monitoring synergistic effects of gene depletion on the activity of mitochondria-specific DNA-damaging agents, we identify a series of proteins not previously recognized to act within mitochondria. These include proteins that function in pathways of oxidative DNA damage repair and dsDNA break repair, along with a novel mitochondrial DNA polymerase, POLθ, that facilitates efficient DNA replication in an environment prone to oxidative stress. POLθ expression levels affect the mutational rate of mitochondrial DNA, but this protein also appears critical for efficient mtDNA replication.

Boronic acids have attracted the attention of synthetic and medicinal chemists due to boron’s ability to modulate enzyme function. Recently, we demonstrated that boron-containing amphoteric building blocks facilitate the discovery of bioactive aminoboronic acids. Herein, we have augmented this capability with a de novo library design and virtural screening platform modified for covalent ligands. This technique has allowed us to rapidly design and identify a series of α-aminoboronic acids as the first inhibitors of human ClpXP, which is responsible for the degradation of misfolded proteins.

We designed a novel, manualized intervention called Emotion And Symptom-focused Engagement (EASE) for acute leukemia (AL) and report here on a phase II randomized controlled trial (RCT) to assess its feasibility and preliminary efficacy.Patients were recruited within 1 month of hospital admission and randomized to EASE plus usual care (UC) or UC alone. EASE includes (1) EASE-psy, a tailored psychotherapy delivered over 8 weeks, and (2) EASE-phys, weekly physical symptom screening over 8 weeks to trigger early palliative care. The primary outcome was traumatic stress symptoms; secondary outcomes included physical symptom burden and quality of life. Assessments were conducted at baseline and at 4, 8, and 12 weeks. Between-group differences were evaluated using multilevel modeling.Forty-two patients were randomized to EASE (n = 22) or UC (n = 20), with 76% retention at 12 weeks. Predefined feasibility outcomes were met: 86% (19/22) of EASE participants completed ≥ 50% of EASE-psy sessions (goal ≥ 64%); 100% received Edmonton Symptom Assessment System (ESAS, modified for AL) screenings, 64% (14/22) of whom completed ≥ 50% of planned screenings (goal ≥50%); and 100% with scores ≥ 4/10 on any physical ESAS-AL item had ≥ 1 meeting with the EASE-phys team (goal 100%). Significant treatment-group differences favoring EASE were observed in traumatic stress symptoms at 4 and 12 weeks, and pain intensity and interference at 12 weeks (all p .05).EASE is feasible in patients newly diagnosed with AL and shows promise of effectiveness. These results warrant a larger RCT to provide evidence for its more routine use as a standard of care.

Mitochondria possess unique proteases that localize to specific sub-compartments of the organelle. However, the functions of these proteases are largely ill-defined. Here, we used proximity-dependent biotinylation (BioID) to map the interactomes of seven proteases located in the intermembrane space of the mitochondria. The mitochondrial intermembrane space proteases HTRA2, OMA1, YME1L1, LACTB, IMMP1L, IMMP2L and PARL were cloned in-frame with the abortive E. coli biotin ligase BirA*, and expressed in 293 T-REx cells. Cell culture media was spiked with biotin for 24 hrs, the cells lysed, and biotinylated proteins were isolated and identified by mass spectrometry. In total, we identified 342 different proteins as high confidence interactors of the seven mitochondrial proteases. Of these, 272 are assigned a GO mitochondrial annotation, and 230 proteins interacted with only 1 or 2 proteases in our dataset. Validation efforts were focused on high temperature requirement peptidase A 2 (HTRA2). HTRA2 is a serine protease that is released into the cytoplasm during apoptosis where it binds Inhibitor of Apoptosis Proteins (IAPs). However, little is known about the function of HTRA2 in the mitochondria. HTRA2 interacted with 60 mitochondrial, 11 nuclear and 4 cytoplasmic proteins, including its known interactor XIAP, and consistent with its known localization to these cellular compartments. HTRA2 interacted with 8 out of 13 components of the MIB complex, a multiprotein assembly that is essential for proper mitochondrial cristae formation. Knockdown of HTRA2 with shRNA in 293T-REx cells disrupted cristae formation and this phenotype was rescued by expression of an shRNA-resistant HTRA2 cDNA. Compared to normal hematopoietic cells, HTRA2 mRNA expression levels are increased in a subgroup of primary AML cells. HTRA2 knockdown in OCI-AML2 leukemia cells led to a similar disruption of mitochondrial cristae. Knockdown of HTRA2 in OCI-AML2 cells led to increased levels of the MIB subunit IMMT, but not two other MIB complex subunits, SAMM50 and CHCHD3. Finally, in cell-free assays, we demonstrate that recombinant HTRA2 can degrade recombinant IMMT, but not SAMM50 or CHCHD3.Thus, we have mapped the interactomes of the proteases of the mitochondrial intermembrane space. Through this effort, we discovered that HTRA2 regulates protein levels of the MIB complex subunit IMMT and that disruption of this process affects mitochondrial cristae formation. Citation Format: Aaron D. Botham, Etienne Coyaud, Sanjit Nirmalanandhan, Marcela Gronda, Rose Hurren, Neil Maclean, Jonathan St. Germain, Sara Mirali, Estelle Laurent, Brian Raught, Aaron Schimmer. Mapping the protein interactome of mitochondrial intermembrane space proteases identifies a novel function for HTRA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4529.

We analyzed the outcome of 80 sequential patients with core binding factor acute myeloid leukemia and evaluated the influence of molecular monitoring by quantitative reverse transcriptase polymerase chain reaction. With a median follow-up of 5 years, the estimated 5-year relapse-free survival and overall survival were 58% and 66%, respectively. Patients who were in molecular remission at the completion of consolidation chemotherapy had a 21% risk of relapse, while the relapse risk for those in molecular remission at the end of 2 years was 5.5%. Our data indicate that postconsolidation molecular remission does not necessarily preclude disease relapse and further monitoring is required. In contrast, molecular negativity by quantitative reverse transcriptase polymerase chain reaction at the end of 2 years is associated with a low risk of relapse.

Few studies describe the comprehensive immunophenotypic pattern of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in the bone marrow and its treatment. This retrospective analysis evaluates the diagnostic flow cytometry (FCM) pattern and outcome of nine patients diagnosed with BPDCN. A four-tube 10-color FCM panel used for diagnosis of acute leukemia (AL), showed cells in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA-DR, CD71. Seven patients received front-line induction therapy with HyperCVAD with an overall response rate of 86%. Five of six responders underwent planned allogeneic hematopoietic cell transplantation (allo-HCT). For a median follow up of 13.3 months, the 1-year disease free survival and overall survival were 56 and 67%, respectively. An accurate diagnosis of BPDCN can be made by 10-color FCM using a four-tube AL panel demonstrating a characteristic pattern of antigen expression. Front-line induction chemotherapy with HyperCVAD can yield high remission rates, but allo-HCT is required for long-term durable remissions.

Acute leukemia (AL) is a life-threatening cancer associated with substantial morbidity and mortality, particularly in older adults. Given that there has been little research on the psychological impact of such malignancies with acute onset, we assessed the prevalence and correlates of depression and hopelessness in patients with AL.Three hundred forty-one participants were recruited within 1 month of diagnosis or relapse and completed the Beck Depression Inventory-II (BDI-II), Beck Hopelessness Scale (BHS), Memorial Symptom Assessment Scale, and other psychosocial measures. Multivariate regression analyses identified correlates of depression and hopelessness.17.8% reported clinically significant depressive symptoms (BDI-II ≥ 15), 40.4% of which were in the moderate-severe range (BDI-II ≥ 20). 8.5% reported significant symptoms of hopelessness (BHS ≥ 8). Depression was associated with greater physical symptom burden (adjusted R(2) = 48.4%), while hopelessness was associated with older age and lower self-esteem (adjusted R(2) = 45.4%). Both were associated with poorer spiritual well-being.Clinically significant depressive symptoms were common early in the course of AL and related to physical symptom burden. Hopelessness was less common and associated with older age and lower self-esteem. The results suggest that whereas depression in AL may be related to disease burden, the preservation of hope may be linked to individual resilience, life stage, and realistic prognosis.Copyright © 2015 John WileySons, Ltd.

Acute leukemia is a condition with an acute onset that is associated with considerable morbidity and mortality. However, the psychological impact of this life-threatening condition and its intensive treatment has not been systematically examined. In the present study, we investigate the prevalence and correlates of post-traumatic stress symptoms in this population.Patients with acute myeloid, lymphocytic, and promyelocytic leukemia who were newly diagnosed, recently relapsed, or treatment failures were recruited at a comprehensive cancer center in Toronto, Canada. Participants completed the Stanford Acute Stress Reaction Questionnaire, Memorial Symptom Assessment Scale, CARES Medical Interaction Subscale, and other psychosocial measures. A multivariate regression analysis was used to assess independent predictors of post-traumatic stress symptoms.Of the 205 participants, 58% were male, mean age was 50.1 ± 15.4 years, 86% were recently diagnosed, and 94% were receiving active treatment. The mean Stanford Acute Stress Reaction Questionnaire score was 30.2 ± 22.5, with 27 of 200 (14%) patients meeting criteria for acute stress disorder and 36 (18%) for subsyndromal acute stress disorder. Post-traumatic stress symptoms were associated with more physical symptoms, physical symptom distress, attachment anxiety, and perceived difficulty communicating with health-care providers, and poorer spiritual well-being (all p0.05).The present study demonstrates that clinically significant symptoms of traumatic stress are common in acute leukemia and are linked to the degree of physical suffering, to satisfaction with relationships with health-care providers, and with individual psychological characteristics. Longitudinal study is needed to determine the natural history, but these findings suggest that intervention may be indicated to alleviate or prevent traumatic stress in this population.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational conjugation of NEDD8 onto target proteins. Cullin proteins are major substrates of the neddylation pathway, and are involved in diverse cellular processes. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924 in leukemic cells. MLN4924 shows potent activity in K562 human leukemia cells (EC50 = 100 nM). In contrast, by exposing K562 cells to increasing concentrations of MLN4924 over 6 months, we selected a population of cells resistant to MLN4924 (R-K562MLN; EC50 > 2 µM). R-K562MLN cells proliferated similarly to parental K562 cells, and remained resistant to MLN4924 even after culturing in the absence of MLN4924 for 5 weeks. Compared to parental cells, R-K562MLN cells were similarly sensitive to a broad spectrum of chemotherapeutic drugs, including daunorubicin, a classical substrate of the multidrug efflux transporter P-glycoprotein. Thus, the mechanism of resistance to MLN4924 did not appear to be related to generalized multidrug efflux. While basal levels of NEDD8-conjugated cullin proteins were equivalent in parental and resistant cells, NEDD8-cullins were diminished in parental cells treated with 25 nM MLN4924. In contrast, no appreciable reduction in NEDD8-cullins was observed in R-K562MLN cells after treatment with up to 250 nM MLN4924. However, knockdown of NEDD8 by shRNA was cytotoxic to R-K562MLNcells, suggesting that NEDD8 conjugation remains essential for the survival of resistant cells. To further probe mechanisms of MLN4924 resistance in these cells, we sequenced the coding region of the NAE catalytic subunit UBA3 from K562 and R-K562MLN cells, and identified a mutation in codon 310 [ATT (Isoleucine, I) > AAT (Asparagine, N)] in R-K562MLN cells. Biochemical analyses using recombinant wild-type and I310N NAE complexes indicated that the I310N mutation alters biochemical properties of the NAE, increasing the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. The I310N NAE complex was ∼7-fold less sensitive to MLN4924 (IC50 = 225 nM) compared to wild-type NAE (IC50 = 32 nM), providing a mechanistic basis for MLN4924 resistance in R-K562MLNcells through the UBA3 I310N mutation. To investigate whether another malignant leukemic cell line employs a similar on-target resistance mechanism, we similarly selected a population of U937 cells resistant to MLN4924 (R-U937MLN). Parental U937 cells were sensitive to MLN4924 (EC50 = 25 nM), while R-U937MLN cells were more than 40-fold more resistant (EC50 > 1 µM). Sequencing of the coding region of UBA3 in U937 and R-U937MLN cells revealed a point mutation in codon 352 [TAT (Tyrosine, Y) > CAT (Histidine, H)] only in R-U937MLN cells. As for the UBA3 I301N mutation, the Y352H mutation conferred a ∼10-fold decrease in the sensitivity of the NAE to MLN4924 (IC50= 138.5 nM and 13 nM for Y352H and wild-type, respectively) through decreased NEDD8 and increased ATP affinities. These findings suggest that the Y352H mutation, like I310N, is sufficient to provide MLN4924 resistance in leukemia cells while sustaining adequate NAE function for leukemic cell survival. We next found that R-K562MLN cells were cross-resistant to other NAE-selective inhibitors derived from Compound 1, a pan-E1 inhibitor. Nevertheless, compared to parental cells, R-K562MLN cells were not refractory to Compound 1 (EC50 = 27 nM and 81 nM, respectively). The cytotoxicity of Compound 1 towards MLN4924-resistant cells might be explained by its inhibition of other E1 enzymes. Indeed, Compound 1 diminished the abundance of ubiquitinated proteins in R-K562MLNcells similarly to its effects in K562 cells, while NEDD8-cullins in the MLN4924-resistant cells were unaffected. Overall, as MLN4924 continues to be evaluated clinically, our work provides insight into the mechanisms of MLN4924 resistance, which may facilitate the development of more effective second-generation NAE inhibitors. Disclosures: Petroski: Allostere, Inc.: Consultancy, Equity Ownership.

Proteasome inhibitors (PIs), such as bortezomib and carfilzomib, are often associated with serious toxicities, poor pharmacokinetics (PK), and the inconvenience of intravenous administration. Therefore, there is a need for safer and more effective orally active PIs. Applying a novel fluorine-based medicinal chemistry technology, we designed, synthesized and tested novel proprietary analogs of epoxyketone-based PIs. Our initial screening identified a lead orally bioavailable PI, FV-162, which had superior potency in proteasome activity inhibition, cytotoxicity in cultured human multiple myeloma (MM) cells and metabolic stability in mouse liver microsomes, compared to an oral PI currently under clinical evaluation, ONX-0912. FV-162 inhibited the β subunits of purified proteasomes of T. acidophilum and specifically inhibited the β5 subunit of the proteasome in 10 human myeloma cell lines with an IC50 range of 12-58 nM. In comparison, IC50 values for the β1 and β2 subunits were consistently greater than 10 µM. FV-162 also disrupted the ubiquitin-proteasome pathway in the LP-1 human myeloma cell line in a time- and dose-dependent manner, inducing intracellular accumulation of ubiquitylated proteins. FV-162 was cytotoxic to 10 human MM cell lines with an IC50 range of 16-611 nM. Furthermore, in 4 MM patient samples tested, at least 80% of primary CD138+ myeloma cells were preferentially killed by FV-162 at concentrations less than or equal to 0.5 µM after 24 hours of incubation, while more than 70% of CD138- normal hematopoietic cells remained viable. FV-162 displayed permeability in murine gastrointestinal epithelial cells, consistent with excellent oral absorption. In addition, cytochrome P450 isozyme assays in pooled human liver microsomes using up to 3 μM FV-162 suggested minimal risk for drug-drug interaction. The toxicity of epoxyketone-based PIs, such as carfilzomib, appears to be related to the Cmax, while efficacy is more dependent on the AUC. We hypothesized that the novel fluorine chemistry of FV-162 would enhance the stability of the drug and thereby improve its PK profile and therapeutic index. Therefore, we measured the oral PK of FV-162 and ONX-0912 in NOD/SCID mice (25 mg/kg). FV-162 reached a peak plasma concentration (Cmax) of 21 ± 5 ng/mL (mean ± SD) after 1 h (Tmax), while ONX-0912 reached a Cmax of 38 ± 46 ng/mL after a Tmax of 5 min. Despite the lower Cmax, the AUC of FV-162 was 6-fold higher compared to ONX-0912 (3117 vs. 513 min*ng/mL). In addition, the oral bioavailability and half-life of FV-162 were 3- and 4.3-fold higher than ONX-0912, respectively. Attenuated Cmax and improved AUC may improve the tolerability profile of FV-162 compared to ONX-0912 and carfilzomib. Supporting this contention, the maximum tolerated dose of ONX-0912 in NOD/SCID mice was 30 mg/kg, while mice tolerated 200 mg/kg FV-162 for 28 days without significant toxicity. Next, we evaluated FV-162 activity in mouse models. Inhibition of β5 subunit activity reached 80% in murine red blood cells within 1 hour of oral administration of FV-162 to NOD/SCID mice (30-100 mg/kg), which was sustained for at least 24 hours. Oral dosing of 100 mg/kg FV-162 achieved similar β5 inhibition as 30 mg/kg ONX-0912. Finally, continuous daily oral administration of FV-162 at 50 or 100 mg/kg significantly inhibited tumor growth in a myeloma MM.1S mouse xenograft model without toxicity and was as effective as intravenous carfilzomib or ONX-0912 administered at their maximal doses. Taken together, our preclinical data suggest that FV-162 has superior PK, an improved safety profile, and high oral potency, identifying it as a promising orally bioavailable PI for myeloma therapy. Further clinical investigation of this novel PI in MM is therefore warranted. Disclosures: Wang: Fluorinov Pharma Inc.: partial salary/fellowship support Other. Dove:Fluorinov Pharma Inc.: Employment. Climie:Fluorinov Pharma Inc.: Employment. O'Neill:Fluorinov Pharma Inc.: Employment. Slassi:Fluorinov Pharma Inc.: Employment.