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Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTKC481 , we identified BTKT316A , a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts. Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

// Ailin Guo 1,* , Pin Lu 1,* , Greg Coffey 2 , Pamela Conley 2 , Anjali Pandey 2 and Y. Lynn Wang 1 1 Department of Pathology, Lymphoma Translational Pathology, University of Chicago, Chicago, IL, USA 2 Portola Pharmaceuticals, Inc., South San Francisco, CA, USA * These authors have contributed equally to this study Correspondence to: Y. Lynn Wang, email: // Keywords : CLL, cerdulatinib, SYK, JAK-STAT, molecularly targeted therapy Received : December 21, 2016 Accepted : January 01, 2017 Published : January 10, 2017 Abstract Ibrutinib (BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Notably, cerdulatinib blocked proliferation of ibrutinib-resistant primary CLL cells and of BTK C481S -transfected/ibrutinib-resistant lymphoma cells. These anti-tumor effects are well correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition.

Patients with IBD frequently have hematologic abnormalities suggestive of JAK2 mutated MPNs, but are traditionally classified as reactive processes. Haplotype 46/1 is a well-characterized genetic predisposition, common to both inflammatory bowel disease (IBD) and myeloproliferative neoplasms (MPN). In view of this shared genetic predisposition, we measured the frequency of the JAK2V617F mutation in IBD patients with thrombocytosis or erythrocytosis, in order to ascertain whether a higher than expected proportion of these patients may in fact have underlying MPNs. 1121 patients were identified with an active diagnosis of Crohn’s disease or ulcerative colitis, of which 474 had either thrombocytosis or erythrocytosis. Patients with abnormal counts were tested for the JAK2V617F mutation during routine follow-up visits. Interim analysis of first 23 patients tested was performed to assess whether the JAK2V617F positivity rate was statistically significant compared with known expected frequencies in a comparable control population. Of 23 patients, 13 patients had thrombocytosis and 10 had erythrocytosis. Three patients with thrombocytosis (23%), and 1 patient with erythrocytosis (10%), tested positive for JAK2V617F, exceeding the expected thresholds for statistical significance. In patients with IBD and thrombocytosis or erythrocytosis, a meaningful proportion may harbor an undiagnosed MPN, as indicated by clonal abnormalities such as JAK2V617F. These findings imply the need for increased testing of these patients for clonal hematologic abnormalities, and importantly, if found, suggest the need for therapeutic strategies with drugs, such as JAK2 inhibitors, in patients with both MPN and IBD.

Background Although reduction in the JAK2 V617F allele burden (% V617F ) has been suggested as a criterion for defining disease response to cytoreductive therapy in polycythemia vera, its value as a response monitor is unclear. The purpose of this study is to determine whether a reduction in % V617F in polycythemia vera is a prerequisite to achieving hematologic remission in response to cytoreductive therapy. Design and Methods We compared the clinical and hematologic responses to change in % V617F (molecular response) in 73 patients with polycythemia vera treated with either interferon (rIFNα-2b: 28, Peg-rIFNα-2a: 18) or non-interferon drugs (n=27), which included hydroxyurea (n=8), imatinib (n=12), dasatinib (n=5), busulfan (n=1), and radioactive phosphorus (n=1). Hematologic response evaluation employed Polycythemia Vera Study Group criteria, and molecular response evaluation, European Leukemia Net criteria. Results Of the 46 treated with interferon, 41 (89.1%) had a hematologic response, whereas only 7 (15.2%) had a partial molecular response. Of the 27 who received non-interferon treatments, 16 (59.3%) had a hematologic response, but only 2 (7.4%) had a molecular response. Median duration of follow up was 2.8 years. Statistical agreement between hematologic response and molecular response was poor in all treatment groups. Conclusions Generally, hematologic response was not accompanied by molecular response. Therefore, a quantitative change in % V617F is not required for clinical response in patients with polycythemia vera.

Ibrutinib, a Bruton's tyrosine kinase inhibitor, is active in CLL, but resistance may emerge. The authors observed the emergence of a CLL clone with a cysteine-to-serine change in amino acid 481 of the target protein that substantially weakens drug binding and leads to resistance.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Chronic myeloproliferative neoplasms (MPNs) are a group of related conditions characterized by the overproduction of cells from one or more myeloid lineages. More than 95% of cases of polycythemia vera, and roughly half of essential thrombocythemia and primary myelofibrosis acquire a unique somatic 1849G>T JAK2 mutation (encoding V617F) that is believed to be a critical driver of excess proliferation. We report here that JAK2(V617F)-associated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (polycythemia vera, n = 192, P = 2.9 x 10(-16); essential thrombocythemia, n = 78, P = 8.2 x 10(-9) and myelofibrosis, n = 41, P = 8.0 x 10(-5)). Furthermore, JAK2(V617F) specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of JAK2(V617F)-associated MPNs (OR = 3.7; 95% CI = 3.1-4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation.

In chronic lymphocytic leukemia (CLL), patients with unmutated immunoglobulin heavy chain variable region gene (UM-CLL) have worse outcomes than mutated CLL (M-CLL) following chemotherapy or chemoimmunotherapy. However, in the era of BCR-targeted therapies, the adverse prognostic impact of unmutated IGHV seems to be diminishing, and there are clinical datasets showing unexpected improved responses in UM-CLL. We investigated the biological differences of BTK activity between these subgroups and further compared the impact of ibrutinib on molecular and cellular behaviors. Immunoblotting analysis revealed that phosphorylated active BTK is significantly higher in UM-CLL. Moreover, UM-CLL, compared to M-CLL, displayed a much higher proliferative capacity that was correlated with higher phospho-BTK and greater sensitivity to ibrutinib. In addition, BTK depletion with siRNA led to a more prominent reduction in the proliferation of UM-CLL, suggesting that elevated BTK activity is responsible for increased cell proliferation. Further, cell signaling activity by multiple measurements was consistently higher in UM-CLL accompanied by a higher sensitivity to ibrutinib. These studies link UM-CLL to elevated BCR signaling, heightened BTK-dependent cell proliferation and increased sensitivity to ibrutinib. The prognostic significance of IGHV mutation should be reevaluated in the era of new therapies targeting BCR signaling.

Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

Monitoring breakpoint cluster region-Abelson kinase (BCR-ABL) levels in patients treated for chronic myelogenous leukemia (CML) has become an integral part of patient management. Real-time reverse transcriptase- polymerase chain reaction is the method of choice for this purpose because of its high analytical sensitivity and reproducibility. Given the variation of RNA quality and quantity in clinical specimens, accurate quantitative assessment of BCR-ABL depends on normalization of the BCR-ABL signal to an appropriate internal reference. However, the controls used by different laboratories vary, and there is no clear consensus on an ideal reference due to limited investigations. In this study, we compared nine commonly used control genes for three criteria: mRNA abundance, levels in CML and non-CML cells, and their degradation kinetics in comparison with BCR-ABL. We found that β-glucuronidase (GUSB) is the most suitable among the nine genes tested. Although ABL is most widely used, our data suggest that the amount of ABL is different in CML and non-CML cells. Moreover, ABL levels are regulated by cellular stress. These findings have a direct impact on current clinical laboratory practice and patient care because the use of a proper control gene affects the reported levels of BCR-ABL transcripts used for patient management decisions.

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach.

An international basis for comparison of BCR-ABL mRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABL values generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABL difference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.2-fold as achieved by 89% of methods, there was good agreement between the overall MMR rates. This indicates that the IS can deliver accurate comparison of molecular response rates between clinical trials when measured by different laboratories.

Introduction Ibrutinib (ibr), a first-in-class BTK inhibitor, has high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL) independent of high-risk FISH abnormalities (NEJM. 2015; 373:2425-37, NEJM. 2013; 369:32-42). However, about 25% of patients discontinue ibr therapy at a median of 20 months treatment and ~40% patients stop ibr due to disease progression (JAMA Oncol 2015; 1:80-7, Blood 2015; 125:2062-67). Among progressed patients, at least half developed Richter's transformation (RT). Treatment options following progression are limited, with mortality rates exceeding 75% and a short median overall survival of 3 months. As the use of ibr becomes more prevalent in CLL and other types of non-Hodgkin lymphoma (NHL), more patients are at risk to develop resistance (BJH 2015; 170:445-56). Strategies to prevent and treat ibr-relapsed patients by understanding mechanisms of resistance are critically needed and will support rational drug development and therapeutic approaches. Recent studies including ours have provided some insights into ibr-resistance. Both BTK and PLCG2 mutations have been shown to confer ibr-resistance (NEJM 2014; 370:2352-54, NEJM 2014; 370:2286-94). Additionally, TRAIL-R has also been associated with the drug resistance in several ibr-relapsed patients (Nat Comm 2016). However, there is still limited understanding in the setting of disease progression during ibr treatment, such as what risk factors predispose patients to relapse, whether other molecular or cytogenetic lesions are associated with disease progression, and how RT is related to CLL tumor cells in the blood. Materials & Methods To address these questions, we analyzed 9 CLL patients treated with ibr and relapsed at the University of Chicago between 2008-2016. Among 9 patients, 6 developed RT at progression. The median age was 66.3 yrs (range, 52-88) and the median number of therapies prior to ibr initiation was 2 (range, 1- 4). The median duration of response to ibr was 16 months (range, 2-30). Eight patients discontinued ibr therapy due to CLL progression or RT. Longitudinal samples including peripheral blood (PB), bone marrow (BM) and tissue were collected from each patient at time points prior to ibr initiation (pre-ibr), post-relapse and at the time of RT. When possible, samples were also collected during the responding phase. Samples were analyzed using both UCM-OncoPlus, a hybrid capture 1,212 cancer-associated Next generation sequencing (NGS) gene panel and Affymetrix SNP arrays (CytoScan and OncoScan) to assess mutations, indels, copy number variations (CNVs) and loss of heterozygosity. A custom algorithm was developed to calculate mutant clonal frequencies (MCFs) using an integrative approach combining allelic frequencies, tumor purity and CNV data. K-means clustering was performed to identify gene mutations belonging to the same clonal populations. An amplicon-based 17-gene CLL panel was further used to sequence BTK in greater depth (~10,000x) to confirm the presence of minor clones (1-5%) in some samples. Results & Conclusions To determine the risk factors associated with relapse, we compared all pre-ibr samples for recurrent molecular and cytogenetic abnormalities. Eight of nine patients were found to have TP53 mutations and/or del (17p), several other genetic lesions also seem to be enriched in our cohort compared to larger populations of treated or untreated CLL patients. The mutation profiles of matched pre-ibr and relapsed CLL PB/BM for each patient were then compared to identify relapse-specific alterations that might drive CLL progression. As expected, BTK mutations were found in 5 of 9 patients including previously reported C481S/R as well as a structurally novel T316A located in the SH2 domain. The findings confirm that mutated BTK is the most common mechanism responsible for disease progression on ibr. Furthermore, the emergence of minor BTK clones was detected in progressed patients. Analyses are ongoing to determine the clonal relationship between CLL leukemia in PB/BM and large cell transformation at the tissue site. So far, IGH gene rearrangement assay on three of five patients demonstrated that the CLL and RT are of the same B-cell origin. We are also in the process of performing cluster analyses to understand mutations that travel in the same malignant clones or sub-clones in each patient and updated results will be presented. Disclosures Smith: Celgene: Consultancy; Amgen: Other: Educational lecture to sales force; Genentech: Consultancy, Other: on a DSMB for two trials ; AbbVie: Consultancy; TGTX: Consultancy; Gilead: Consultancy; Portola: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy. Wang:Portola Pharmaceuticals: Honoraria, Research Funding.

Abnormal upregulation of NFκB activity is observed in a variety of B cell malignancies, resulting in proliferative and survival signals that contribute to tumor progression. Under normal resting conditions, NFκB is negatively regulated principally via its physical association with IκB (inhibitor of NFκB) family members, thereby inhibiting nuclear transport or access to DNA. In B cells, NFκB is typically activated via various external stimuli (e.g., ligation of the B cell antigen receptor (BCR), toll-like receptors, cytokine receptors, CD40), leading to IκB kinase complex-dependent phosphorylation of IκB members and targeting them for ubiquitination and degradation. In some cases, the need for external stimuli is diminished or completely circumvented by mutations to critical regulators of NFκB, as has been described in the context of activating mutations to CD79A/B, MYD88, and CARD11, as well as inactivation of negative regulators such as A20 and IκB family members (reviewed by Staudt, 2010). Each of these mutations has been observed clinically in patients with B cell malignancies (Wilson et al, 2012; Norenberg et al, 2015; Mansouri et al, 2015), and can impact the anti-tumor activity of selective BCR pathway inhibition (Davis et al, 2010; Wilson et al, 2012) in part via induction of autocrine cytokine stimulation leading to JAK/STAT-dependent up-regulation of MCL1 (Lam et al, 2008). We previously reported that cerdulatinib, a small molecule kinase inhibitor that dually targets SYK and the JAK family members JAK1, JAK3, and TYK2, maintained anti-tumor activity in DLBCL cell lines bearing mutations to CARD11, MYD88, and A20 (Ma et al, 2015). The majority of DLBCL cell lines exhibit various degrees of reliance on SYK and JAK signaling for survival, however in a screen of 15 DLBCL cell lines we found 3 that were completely resistant to cerdulatinib and are described here. In one of the cerdulatinib-resistant cell lines, RCK8, next generation sequencing revealed bi-allelic inactivation of the IκBα gene. One allele carries a frameshift mutation in exon 1 resulting in the generation of a premature stop codon, and the second allele is a nonsense mutation in exon 3 at Gln154, also leading to a stop codon. In accord with a previous report (Kalaitzidis et al, 2002), the cell line lacks IκBα protein expression. We therefore proceeded to explore the possibility that loss of IκBα was responsible for resistance to cerdulatinib. Consistent with the loss of IκBα, the RCK8 cell line exhibited enhanced basal NFκB activity. Genetic re-introduction of wild type IκBα led to rapid suppression of NFκB, and ultimately cell cycle arrest and cell death, indicating that the cell line was dependent upon loss of this gene for survival. Associated with suppression of NFκB was decreased phosphorylation of cellular pAKT S473 and pERK Y202, but not of pSTAT3 Y705. We then attempted to knock down IκBα in cerdulatinib-sensitive cell lines using siRNA to determine if resistance to SYK/JAK inhibition could be generated. None of the DLBCL cell lines tested (n=4) could tolerate IκBα gene knock down, suggesting an additional mutation in RCK8 enables survival under conditions of homozygous loss of IκBα. Ligation of CD40 leads to a transient down-regulation of IκBα at the protein level (Oeckinghaus and Ghosh, 2009). We therefore examined the effect of CD40L on multiple DLBCL cell lines and found that IκBα was maximally suppressed within 30-60 minutes post CD40 stimulation, returning to pre-treatment levels by 2-4 hours. In contrast, the impact on NFκB activation was much longer, and 5 of 7 cerdulatinib-sensitive cell lines tested were made resistant by incubation with CD40L. Associated with this resistance was not only induction of NFκB, but also pERK Y204, pAKT S473, and pSTAT3 Y705. Interestingly, whereas the CD40L-induced NFκB activation was not inhibited by cerdulatinib, the other signaling events were, despite the generation of resistance. Loss of IkB family members has been described in the context of Hodgkin's lymphoma, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia (Cabannes et al, 1999; Norenberg et al, 2015; Mansouri et al, 2015). Here we demonstrate that loss of IκBα in multiple DLBCL cell lines generates resistance to cerdulatinib. We will be exploring the clinical relevance of these in vitro observations in cell lines as part of an ongoing phase II trial of cerdulatinib in patients with relapsed/refractory B cell malignancies. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Feng:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Wang:Portola Pharmaceuticals: Honoraria, Research Funding. Michelson:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Curnutte:3-V Biosciences: Equity Ownership; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding; Sea Lane Biotechnologies: Consultancy. Conley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

In a series of 105 patients with polycythemia vera, we retrospectively determined whether the JAK2(V617F) mutation correlated with severity of disease phenotype. Higher JAK2(V617F) allele burden correlated with more advanced myelofibrosis, greater splenomegaly, and higher white blood cell count, but not with age, gender, hematocrit level, or frequency of phlebotomy prior to cytoreductive therapy. Although a subgroup at increased risk for thrombosis was not clearly defined, there was a suggestion that frequency of thrombosis increased as the JAK2(V617F) allele burden increased. The JAK2(V617F) allele burden did not change significantly in treated patients with serial JAK2 analyses.

The M-protein is the major reference measure for response in multiple myeloma (MM) and its correct interpretation is key to clinical management. The emergence of oligoclonal banding is recognized as a benign finding in the post-autologous stem cell transplantation setting (ASCT) for MM but its significance during non-myeloablative therapy is unknown. In a study of the immunomodulatory combination BiRD, (lenalidomide and dexamethasone with clarithromycin), we frequently detected the emergence of mono- and oligo-clonal immunoglobulins unrelated to the baseline diagnostic M-protein. The new M-proteins seen on serum immunofixation electrophoresis were clearly different in either heavy or light chain component(s) from the original M-spike protein and were called atypical serum immunofixation patterns (ASIPs). Overall, 24/72 (33%) patients treated with BiRD developed ASIPs. Patients who developed ASIPs compared with patients treated with BiRD without ASIPs, had a significantly greater overall response (100% vs. 85%) and complete response rates (71% vs. 23%). ASIPs were not associated with new clonal plasma cells or other lymphoproliferative processes, and molecular remissions were documented. This is the first time this phenomenon has been seen with regularity in non-myeloablative therapy for MM. Analogous to the ASCT experience, ASIPs do not signal incipient disease progression, but rather herald robust response.

Importance Next-generation sequencing has identified new genetic markers that have altered prognosis for patients with chronic lymphocytic leukemia (CLL) at diagnosis. Understanding the significance of these prognostic indicators and recognizing their potential impact on treatment selection and patients' outcomes is critical for clinicians and investigators. Objective To review novel prognostic factors at CLL diagnosis that have shown an impact on the prognosis and outcomes of this disease. Evidence review Literature from January 2004 through December 2014 was searched in PubMed, Cochrane Central Register of Controlled Trials, and Scopus to identify English-language, peer-reviewed articles on clinical and prognostic factors for CLL (TP53, ATM, NOTCH1, SF3B1, BIRC3, and MYD88). Reference lists were subsequently reviewed for additional articles. A total of 450 articles was identified, and 48 articles meeting inclusion criteria were reviewed. Findings Among prognostic markers reviewed, chromosomal aberrations have been validated and are currently used clinically to predict prognosis. Patients with 17p13.1 deletion have poor response to chemoimmunotherapy and are treated differently, with some undergoing allogeneic transplantation in first remission. CD38 and ZAP-70 status of malignant cells and unmutated immunoglobulin variable heavy chain gene have similarly been validated to predict adverse prognosis, but their implications on treatment selection have not been proven. The presence of TP53 and ATM mutations predicts worse prognosis, which has been corroborated in various studies. Patients with TP53 mutations have lower responses to chemoimmunotherapy. Furthermore, patients with TP53 and ATM mutations have inferior progression-free survival and overall survival, independent of other factors. Patients carrying the NOTCH1 and SF3B1 mutations have worse prognosis; patients with the NOTCH1 mutation have lower response rates to standard chemoimmunotherapy. The impact of BIRC3 on prognosis and survival requires further confirmation. Conclusions and relevance The heterogeneous clinical course of CLL is likely explained by underlying molecular prognostic factors. Moving forward, analyzing these factors at diagnosis is recommended for better prognostication.

Peroxisome proliferator-activated receptor γ (PPARγ) is a metabolic regulator that plays an important role in sensitizing tissues to the action of insulin and in normalizing serum glucose and free fatty acids in type 2 diabetic patients. The receptor has also been implicated in the modulation of inflammatory responses, and ligands of PPARγ have been found to induce apoptosis in lymphocytes. However, apoptosis induction may not depend on the receptor, because high doses of PPARγ agonists are required for this process. Using cells containing or lacking PPARγ, we reported previously that PPARγ attenuates apoptosis induced by cytokine withdrawal in a murine lymphocytic cell line via a receptor-dependent mechanism. PPARγ exerts this effect by enhancing the ability of cells to maintain their mitochondrial membrane potential during cytokine deprivation. In this report, we demonstrate that activation of PPARγ also protects cells from serum starvation-induced apoptosis in human T lymphoma cell lines. Furthermore, we show that the survival effect of PPARγ is mediated through its actions on cellular metabolic activities. In cytokine-deprived cells, PPARγ attenuates the decline in ATP level and suppresses accumulation of reactive oxygen species (ROS). Moreover, PPARγ regulates ROS through its coordinated transcriptional control of proteins and enzymes involved in ROS scavenging, including uncoupling protein 2, catalase, and copper zinc superoxide dismutase. Our studies identify cell survival promotion as a novel activity of PPARγ and suggest that PPARγ may modulate cytokine withdrawal-induced activated T cell death.

The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib has produced durable remissions in chronic lymphocytic leukemia (CLL). We describe a CLL patient who progressed while receiving ibrutinib following 20 months of once daily dosing. A cysteine-to-serine amino acid replacement was identified in BTK at position 481 that disrupts the covalent, but not non-covalent, binding of ibrutinib to BTK in silico structural modeling1. The mutation was present in relapsed samples while absent in the pre-treatment and responding samples. Following the mutation, the B cell receptor (BCR) pathway was reactivated as evidenced by increased cell signaling activities and gene expression profiles. Comparing the relapsed samples with the pre-treatment and responding samples, at the cellular level, mutated CLL cells displayed higher levels of the cell proliferation marker Ki67 in vivo and higher levels of ex-vivo BrdU incorporation. Transfection of the C481S mutant construct into a sensitive lymphoma cell line rendered it much more resistant to ibrutinib treatment demonstrating the cellular impact of the mutation (see attached graph). Interestingly, the ibrutinib-resistant CLL cells remained sensitive to other BCR inhibitors such as dasatinib and SYK inhibitors. These results confirm BTK as an important pharmacologic target of ibrutinib. Further, a mechanism of resistance was revealed, and alternative therapeutic options for ibrutinib resistance were explored. (First three authors with equal contribution) Disclosures: Furman: Genentech: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Gilead: Consultancy. Chang:Pharmacyclics: Employment, Equity Ownership.

With increasing understanding of the genetic basis of diffuse large B cell lymphoma (DLBCL), more targeted therapies have been developed. However, most of these therapies have activities only against a subset of DLBCL, activated B-cell like subtype (ABC), in particular, where the BCR pathway is known to be chronically active. Specific single-molecule directed therapies, although effective, often induce resistance over long term treatment. Agents with broader activities are needed for the treatment of this heterogeneous disease. Since both the BCR and JAK/STAT pathways are strongly implicated in the pathogenesis of DLBCL, in the present study, we evaluated the anti-lymphoma activity of PRT062070 (PRT2070), a novel compound that dually targets both JAK and SYK signaling pathways. We analyzed a panel of DLBCL cells lines, of both ABC and germinal center B-cell like (GCB) subtypes, as well as DLBCL patient samples, for cellular and molecular events affected by PRT2070. Immunoblotting analyses showed that ABC and GCB subtype of DLBCL cells exhibit different JAK/STAT and BCR signaling profiles. For instance, AKT was highly expressed in GCB cells, whereas STAT3 was more strongly expressed in ABC cells. In GCB cell lines, PRT2070 blocked G1/S transition and caused cell cycle arrest. In contrast, in ABC cells, the drug induced apoptosis and down-regulated MCL1 protein. Furthermore, array of PRT2070-treated ABC subtype of DLBCL cells revealed that several STAT3 regulated cytokines and chemokines, including IL10, were down-regulated, confirming that PRT2070 affects ABC-DLBCL cells via JAK/STAT pathway. Genetic knockdown of both JAK and SYK reduced the growth and the survival of DLBCL cells more pronouncedly than knockdown of JAK or SYK alone, suggesting the anti-tumor activity of PRT2070 was mediated by dual inhibition of JAK and SYK signaling pathways. Importantly, JAK/STAT and BCR signaling can be blocked by PRT2070 in GCB and non-GCB primary human DLBCL cells, which led to death of these cells. Our work provided mechanistic insights into the actions of JAK/SYK dual inhibitor PRT2070 suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures: Coffey: Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment.

Abstract 5068 Busulfan, a highly effective and established drug in polycythemia vera (PV), produces lasting clinical and hematologic responses. Its use as a first and second line therapy for PV recently diminished owing to largely unsubstantiated concerns of increased leukemogenicity. However, in a pivotal phase III trial of busulfan vs. P32 conducted by the EORTC in patients with PV, busulfan sustained long term clinical and hematologic responses as well as superior 10 year overall survival (70% vs. 55%). Toxicity was minimal and a low incidence of acute leukemia was reported (1% at 8 years). Accordingly, we treated 5 PV patients with busulfan, 4 of whom were refractory to multiple drugs including hydroxyurea (HU), pegylated interferon alfa-2a (pIFN), imatinib, dasatinib, and anagrelide. Clinical and hematologic response was graded according to PVSG criteria and molecular response according to ELN criteria. JAK2V617F allele burdens were determined by pyrosequencing, which quantifies mutant alleles more than 5%. If negative by pyrosequencing, we used an ARMS-PCR assay with a sensitivity of 0. 1%. Phlebotomy was performed to maintain the hematocrit (Hct) less than 45% for men and 42% for women. Treatment with busulfan was discontinued if patients experienced adverse side effects and/or had platelet counts less than 100, 000/mL while in clinical remission. All 5 patients had complete hematologic responses (CHR) within 3 months of starting busulfan (table 1). Molecular responses (MR) were: 1 complete (CMR) after 6 months, 1 partial (PMR) after 6 months, and 3 with no response (NMR) after 3, 7, and 60 months of treatment respectively. The 2 patients who had MR were homozygous for JAK2V617F, and the 3 who did not were heterozygous. Treatment was discontinued in the patient with CHR and CMR after 7 months due to thrombocytopenia; the patient has since maintained CHR and CMR for 3 years off treatment. The remaining 4 patients have maintained CHR on low doses of busulfan (table 1). No adverse events were observed over a median treatment duration of 15 months (range: 3–60 months). The significant difference in molecular response between patients with homozygous and heterozygous JAK2 mutations receiving similar doses of busulfan is noteworthy. This suggests a susceptibility of homozygous JAK2V617F clones to busulfan, but not to other drugs including HU, IFNa, and anagrelide. In summary, our 5 patients with multidrug refractory PV had rapid and sustained hematologic responses to busulfan at low doses, with favorable short and long term toxicity profiles. Two JAK2V617F homozygous patients had the best MR. Our findings indicate the effectiveness of a safe, relatively inexpensive drug in inducing clinical outcomes not significantly different from that of costly drugs, such as JAK2 inhibitors. In addition, the high rates of MR we observed in patients with homozygous JAK2 mutations warrant further study of busulfan with respect to this parameter. Table 1: Demographics and treatment results of 5 patients treated with busulfan for PV Patient Age (yr)-Sex (M/F) Prior treatments-duration (yr) Busulfan dose Adverse effects Duration of tx (months) Hematologic response/time to response (months) Molecular response/time to response (months) Pre-busulfan JAK2 allele burden 1 75-F HU-2 yr 4mg daily thrombocytopenia 7 CHR/3 CMR/6 100% 2 70-F HU+anagrelide-1yr Imatinib-2yr Dasatinib-3 yr IFNa-1 yr 2mg 3 times a week None 15 CHR/1 PMR/6 85% 3 84-F HU-2 yr Dasatinib- 3yr Imatinib-1yr 2mg 4 times a week None 18 CHR/3 None 27% 4 81-M HU-5 yr 2mg daily None 3 CHR/2 None 23% 5 81-F None 2mg 3 times a week None 60 CHR/3 None 45% Disclosures: No relevant conflicts of interest to declare.

Abstract 1249 Poster Board I-271 Purpose B-cell receptor (BCR) signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking BCR signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in pre-clinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. Experimental Design Fresh CLL B cells were treated with dasatinib and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of BCR signaling molecules as well as with molecular and cytogenetic prognostic factors. Results and Conclusions Among 44 CLL cases, dasatinib treatment reduced cell viability by 2-90%, with an average reduction of 48% on day four of culture. The drug induced CLL cell death via the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good as well as poor responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and PLCγ2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with PLCγ2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. Disclosures Lee: Bristol-Myers Squibb Research & Development: Employment. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.

B cell receptor (BCR) signaling plays an essential role in the pathogenesis of chronic lymphocytic leukemia. In a subset of patients with a poor clinical outcome, BCR ligation leads to increased cell metabolism and cell survival (Cancer Research66, 7158–66, 2006). Based on these findings, we tested whether targeting BCR signaling with dasatinib, an inhibitor of Src kinase, would interfere with the signaling cascade and cause death of CLL B cells. CLL leukemic cells were isolated from 34 patients and were incubated with or without dasatinib at a low dose of 128 nM. Among 34 cases, viability of leukemic cells was reduced by 2% to 90%, with an average of ~50% reduction on day 4 of ex vivo culture. Further study showed that CLL B cells undergo death by apoptosis via the intrinsic pathway which involves the generation of reactive oxygen species. Analysis of the Src family kinases showed that phosphorylation of Src, Lyn and Hck was inhibited by dasatinib not only in those cases that responded to dasatinib with apoptosis, but also in those that did not respond well ( Statistical analysis showed a significant correlation between CLL dasatinib response and their IgVH mutation and ZAP70 status. Cases with worse prognoses by these criteria have a better response to the kinase inhibitor. Lastly, we have also found that ZAP70 positive cases showed a greater degree of PLC

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear hormone receptor involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPAR gamma induce apoptosis in several types of tumor cells including lymphomas, leading to the proposal that these ligands may be used as anti-cancer agents. However, apoptosis induction requires high doses of ligands, suggesting the effect may not be receptor-dependent. Previously, we reported that PPAR gamma is expressed in human primary T cell lymphoma tissues and activation of PPAR gamma with low doses of ligands protects lymphoma cells from serum starvation-induced apoptosis. The prosurvival effect of PPAR gamma is linked to its actions on mitochondria. In serum-deprived cells, PPAR gamma attenuates the decline in ATP, reduces mitochondrial hyperpolarization and limits the amount of reactive oxygen species (ROS) in favor of cell survival (Yang et al, Am J Pathol, 170, 722–32, 2007). In the current study, we investigated the molecular mechanisms by which PPAR gamma maintains mitochondria homeostasis. We demonstrated that PPAR gamma modulates the activities of two key components of Wnt signaling pathway, Fzd4 and GSK-3 beta. The receptor up-regulates the mRNA expression of Fzd4, a cell surface receptor of Wnt, through a conserved PPAR-response element (PPRE) in the promoter region of the Fzd4 gene. Moreover, PPAR gamma suppresses the activation of GSK-3 beta, a downstream inhibitory serine/threonine kinase, by maintaining its phosphorylation at serine 9 and decreasing its association with mitochondria. Downregulation of GSK-3 beta activity results in maintenance of mitochondrial membrance potential and suppression of ROS production in growth factor-deprived cells. Our studies revealed a novel interaction between PPAR gamma and Wnt signaling in lymphocyte survival. Since Wnt signaling plays an important role in tumorigenesis, our studies highlight the need for further investigation into the role of PPAR gamma in cancer prior to widespread use of its agonists as anticancer therapeutics.

Somatic hypermutation of immunoglobulin genes (SHM) is a physiological process that helps generate high affinity antibodies in germinal center B cells, and error-prone DNA polymerases are key enzymes involved in this process. Unlike polymerases that are responsible for DNA replication, error-prone DNA polymerases copy DNA with low fidelity leading to variations in immunoglobulin DNA sequences. Chronic lymphocytic leukemia (CLL) has been classified into mutated and unmutated subtypes based on the mutation status of the IGH variable region (IgVH). The latter has been associated with rapid disease progression and unfavorable outcome. Since determination of the IgVH mutations rely on direct sequencing of multiple PCR products and complex data analysis, the assay, although clinically useful, is not provided by many clinical laboratories. To determine whether error-prone DNA polymerases can be used as surrogate markers for IgVH mutation analysis, we examined the levels of several of them to see whether higher levels are associated with higher degree of IgVH mutation. We analyzed 30 CLL samples, of which 18 were unmutated and 12 were mutated. Quantitative PCR was performed to determine the expression levels of error-prone DNA polymerases mu, eta, iota, lambda and zeta. as well as DNA editing enzymes, terminal deoxynucleotidyl transferase (TdT) and activation-induced cytidine deaminase (AID). Statistical analysis revealed no differences in the levels of mRNA expression of any of these seven enzymes between mutated and unmutated cases of CLL. In addition, no differences were found between ZAP-70 positive and negative subgroups. The expression levels of the seven enzymes were then compared among CLL, follicular lymphomas (FL, n=8) and diffuse large B cell lymphomas (DLBCL, n=8). FL and DLBCL, being post-germinal center lymphomas that have undergone SHM, are expected to express higher levels of these enzymes than CLL. However, the statistical analysis revealed that CLL has significantly higher amount of mu, eta, iota, lambda, zeta and TdT expression than DLBCL. CLL also has significantly higher expression of iota, lambda, zeta and TdT than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.

The excess production of monoclonal immunoglobulin is the hallmark of multiple myeloma (MM) diagnosis and is an essential element of the determination of disease response to therapy. The development of new monoclonal immunoglobulins that are distinct from the baseline diagnostic paraprotein during the course of MM therapy can therefore pose a challenge in assessing disease response or potential relapse. There are prior reports of the development of abnormal protein banding (APB) after autologous stem cell transplantation for MM comprised of transient non-myeloma related mono- or oligoclonal protein bands that have been correlated with higher rates of event-free and overall survival. We now report the development of atypical serum immunofixation patterns (ASIPs) for the first time outside of the setting of stem cell transplant during the course of MM induction therapy with the BiRD (Biaxin® [clarithromycin], Revlimid® [lenalidomide], Dexamethasone) regimen. 72 patients with newly diagnosed Stage II and III MM (Salmon-Durie Criteria) were treated in a phase 2 clinical study of BiRD. The BiRD treatment regimen was given in 28-day cycles as follows: Clarithromycin 500mg po BID for days 1 – 28, Lenalidomide 25mg po daily for days 1 – 21, and Dexamethasone 40mg po weekly on days 1, 8, 15, and 21. The median age of the patients was 63 (range 36–83), and the baseline monoclonal immunoglobulins detected on serum immunofixation were as follows: 61% IgG, 22% IgA, 17% light chain only. 24 patients (33%) developed one or more ASIPs with either a mono- or oligoclonal banding pattern during the course of BiRD therapy. The new protein bandsvaried widely in duration of appearance (range 26–315 days) as well as isotype with IgM-κ, IgM-λ, IgG-κ, IgG-λ, IgA-λ, free λ light chain, and free IgM heavy chain all observed in one or more instances. The extent of therapy prior to first ASIP development was variable as well, (range 27– 724 days, median of 178 days). Patients with ASIPs had significantly better response to BiRD vs. non-ASIP patients (p = .00002), with CR+sCR rate of 71% vs. 23%, and a VGPR or better rate of 96% vs. 61%. The extent of BiRD therapy prior to development of first ASIP did not correlate with response rate (p = .7284). Bone marrow examination by histology, karyotype, FISH, and PCR IgH / IgK clonality analysis confirmed the disease response and furthermore showed no evidence for newly emergent plasma cell or other B-cell malignancy to account for ASIP generation suggesting molecular remission in some of the patients. Peripheral blood analysis by flow cytometry also showed no evidence for a circulating B-cell neoplasm to account for the new immunoglobulin production. We propose that ASIP development during myeloma therapy with a lenalidomide-based regimen heralds a robust tumor reduction with a hitherto unprecedented rate of complete remission. The immune phenomena that contribute to ASIP generation are unclear at present, however it is neither due to a new clonal B-cell population or transformation of the original MM plasma cell clone.

Different methods using peripheral blood RNA (Vannucchi AM, et al. Leukemia. 2007,1–8), or archival bone marrow DNA (Tefferi A, et al. Leukemia. 2007,1–2) have yielded varied results correlating allele burden with severity and duration of disease. We therefore aimed to determine whether JAK2V617F allele burden correlated with certain parameters of disease. At our institution, 105 patients were diagnosed according to the criteria of the Polycythemia Vera Study Group. We grouped their JAK2V617F allele burdens into quintiles. DNA from peripheral blood was analyzed using pyrosequencing. For those patients whose allele burden was 9 cm). Thrombotic events were recorded within 5 years of JAK2V617F determination. There were 52 men and 53 women. The patients ranged in age from 35 to 88 years, median 60 years. The median duration of disease was 7.4 years (range: 0.2 - 36.6 years), and the median duration of follow-up after JAK2V617F determination was 12 months year (range: 1 - 43 months). The mean mutant allele burden was 46.0% (s.d. ± 29.7%). The fifth, and highest quintile had a mean mutant allele burden of 90.2% (s.d. ± 5.8%); the lowest quintile had a mean mutant allele burden of 9.9% (s.d. ± 6.3%). JAK2V617F did not correlate with age, gender, hematocrit and platelet count at diagnosis, or rate of phlebotomy prior to cytoreductive therapy. Increasing JAK2V617F burden did correlate with higher WBC at diagnosis (P=0.02), degree of splenomegaly (P

The Bcl-2-associated X protein (Bax) and Bcl-2-antagonist/killer (Bak) are essential regulators of lymphocyte apoptosis, but whether they play a role in viable T cell function remains unclear. Here, we report that T cells lacking both Bax and Bak display defects in antigen-specific proliferation because of Ca(2+)-signaling defects. Bax(-/-), Bak(-/-) T cells displayed defective T cell receptor (TCR)- and inositol-1,4,5-trisphosphate (IP(3))-dependent Ca(2+) mobilization because of altered endoplasmic reticulum (ER) Ca(2+) regulation that was reversed by Bax's reintroduction. The ability of TCR-dependent Ca(2+) signals to stimulate mitochondrial NADH production in excess of that utilized for ATP synthesis was dependent on Bax and Bak. Blunting of Ca(2+)-induced mitochondrial NADH elevation in the absence of Bax and Bak resulted in decreased reactive-oxygen-species production, which was required for T cell proliferation. Together, the data establish that Bax and Bak play an essential role in the control of T cell proliferation by modulating ER Ca(2+) release. ispartof: IMMUNITY vol:27 issue:2 pages:268-280 ispartof: location:United States status: published

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F >50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher’s exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.

Peroxisome proliferator-activated receptor gamma is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPAR gamma have been found to induce apoptosis in a variety of tumor cell lines including lymphomas. However, apoptosis induction may not depend on the receptor since high doses of PPAR gamma agonists are required for this process. Using cells containing or lacking PPAR gamma, we reported previously that PPAR gamma attenuates apoptosis induced by growth factor withdrawal in a murine lymphocytic cell line via a receptor dependent mechanism. PPAR gamma exerts this effect by enhancing ability of cells to maintain their mitochondrial membrane potential during growth factor deprivation. In the current study, we demonstrate that PPAR gamma is expressed in human primary T lymphoma tissues and activation of PPAR gamma protects cells from serum starvation-induced apoptosis in human T lymphoma cell lines. Further, we show that the survival effect of PPAR gamma is mediated through its actions on cellular metabolic activities. In serum-deprived cells, PPAR gamma attenuates the decline in cellular ATP and suppresses accumulation of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPAR gamma regulates ROS through its coordinated transcriptional control of proteins and enzymes involved in ROS production and scavenge. Introduction of PPAR gamma into a PPAR gamma-null T lymphoma cell line leads to increased cell survival. Meanwhile, knocking down the receptor in a PPAR gamma-positive lymphoma cell line reduces cell survival rate. Our studies identify cell survival promotion as a novel activity of PPAR gamma and suggest that high expression of PPAR gamma in lymphoma cells confers on them a survival advantage that renders cells resistant to growth factor and nutrient deprivation. These findings highlight the need for further investigation into the role of PPAR gamma in lymphoma and other types of cancer prior to widespread use of its agonists as anticancer therapeutics.

Abstract 1908 Poster Board I-931 Previous studies of patients (pts) with polycythemia vera (PV) treated with pegylated interferon (peg-IFNá-2a) have shown an 83% complete hematologic response associated with an 89% molecular response over a median of 11 months (Kiladjian et al. Blood. 2008. 112(8):3065-3072) implying a causative relationship between molecular and hematologic responses. Our data show pts treated with rIFNá-2b or non-rIFNá-2b agents achieve hematologic response despite the absence of a molecular response suggesting that a molecular change is not a prerequisite to hematologic response. Thirty pts diagnosed with PV by the criteria of the Polycythemia Vera Study Group (PVSG) were followed clinically and hematologically with serial quantified JAK2V617F allele burden determined at six-month intervals over a mean of 21.6 months (mos) (range: 6.0 – 56.4 mos). These pts were treated with rIFNá-2b ranging from 0.5 mu to 3.0 mu three times per week depending upon clinical response. Primary clinical endpoints were hematocrit (hct) ≤45% men, ≤42% women, and no need for phlebotomy (PHL). Molecular and hematologic responses were graded according to the criteria of Barosi et al. (Blood. 2009. 113(20):4829-4833): complete hematologic response (CHR: hct ≤45% without PHL, platelets '400×109/L, WBC ≤10×109/L, normal spleen size, asymptomatic); partial hematologic response (PHR: hct ≤45% without PHL or response in 3 or more of the CHR categories); no hematologic response (NHR: failure to meet the criteria of CHR or PHR); complete molecular response (CMR: reduction of JAK2V617F marker to undetectable levels); partial molecular response (PMR: ≥50% reduction in pts with '50% mutant allele burden at baseline, or ≥25% reduction in pts with >50% mutant allele burden at baseline; applicable only to pts with ≥10% baseline allele burden); and no molecular response (NMR: failure to meet the criteria of CMR or PMR). Of the 30 pts treated with rIFNá-2b, 14 had a CHR, 13 had a PHR and 3 had NHR. Of 14 pts who had a CHR, 4 had a PMR and 10 had NMR. Of thirteen pts who had a PHR, 1 had a PMR and 12 had NMR. All 3 pts who had NHR also had NMR. Based on these data, the statistical agreement between hematologic response and molecular response was poor (kappa coefficient = 0.06, P=0.17). We then examined the hematologic responses (HR: CHR+PHR) of 25 non-rIFNá-2b treated pts, which included PHL ± anagrelide (3 pts: 2 HR/NMR, 1 NHR/NMR), dasatinib (5 pts: 5 HR/NMR), imatinib (9 pts: 3 HR/PMR, 4 HR/NMR, 2 NHR/NMR), and hydroxyurea (8 pts: 1 CHR/PMR, 7 HR/NMR). The minimal molecular response to dasatinib and hydroxyurea is noteworthy. Likewise, there was poor statistical agreement between hematologic response and molecular response for non-rIFNá-2b treated patients (kappa coefficient = 0.05, P=0.21). Of all 55 pts (rIFNá-2b and non-rIFNá-2b), those 9 patients with a PMR had a hematologic response (7 CHR and 2 PHR). Of 46 NMR's, 40 pts (87%) had a hematologic response (16 CHR, 24 PHR). Thus, NMR did not exclude the possibility of achieving CHR. Regardless of therapy, we demonstrate poor agreement between hematologic and molecular responses for these drugs (all pts: kappa = 0.05, P=0.13). This suggests a difference in action between peg-IFNá-2a, shown to cause molecular and hematologic responses concurrently, and several drugs we examined leading to clinical response without necessarily changing JAK2V617F allele burden. In this regard, other parameters such as bone marrow morphology and new biological markers may be useful in reconciling the differences. In summary, we find that a hematologic response is not always accompanied by a molecular response in PV pts treated with either rIFNá-2b or some non-rIFNá-2b drugs. We thus conclude that a reduction in JAK2V617F allele burden is not always required for patients to achieve hematologic response, and that following the JAK2V617F biomarker may be drug-dependent and may not always be a reliable measure of response. This warrants the importance of the randomized trial planned to compare peg-IFNá-2a to the current standard of treatment, hydroxyurea. Disclosures: No relevant conflicts of interest to declare.

Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways. To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%). SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures Pandey: Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.