ALBERT

Description: Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) receptor plays an important role in the pathogenesis of acute myeloid leukemia (AML). A number of small molecule kinase inhibitors are currently proceeding in different phases of clinical trials. As with imatinib in CML, leukemic cells could develop resistance to these RTK inhibitors when used as monotherapy. Mutations in the ATP-binding pocket have been identified through PCR-based mutagenesis screening in Ba/F3-FLT3-ITD cells and selected for growth in the presence of PKC412, or in a resistant Ba/F3-FLT3-ITD cell line developed by cocultured with SU5416. Resistance to PKC412 resulting from the N676K point mutation in the FLT3 kinase domain has been described in a clinical trial patient. Selection of activating Ras mutations has been found in 2 out of 6 FLT3 inhibitor resistant cell lines, but no point mutation of FLT3 kinase domain was found in all 6 resistant cell lines. To further investigate other potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R (IC50: 52 vs 6 nM for the parental MV4-11 cell line), which is also cross resistant to other structurally unrelated inhibitors including SU5416, AG1296 and a FLT3 inhibitor III (MERCK). No point mutation in the kinase domain of FLT3 was found in MV4-11-R cells. Western blot and FACS analysis excluded overexpression of p-FLT3, FLT3 or 3 multidrug resistance related proteins (MDR, MRP1 and LRP) in this resistant line. Gene expression profiling revealed up-regulation of FLT3 ligand (FLT3LG) (2.4 fold) and Survivin (2 fold), while down-regulation of SOCS1, SOCS2, and SOCS3 was observed in MV4-11-R compared to MV4-11 parental cells. Overexpression of FLT3LG and Survivin was also demonstrated at the protein level. Survivin is a unique member of the inhibitor of apoptosis proteins (IAP) family and a known target of the STAT3 pathway. Down-regulation of suppressor of cytokine signaling (SOCS) proteins (negative regulators of STAT pathways) was observed even in the presence of overactivation of the STAT1, STAT3 and STAT5 pathways in the MV4-11-R line. We screened a panel of small molecule inhibitors including a STAT inhibitor (indirubin derivative IDR E804), 3 JAK inhbitors (Tyrene CR4, AG490, and JAK3 Inhibitor II), and a CDK/survivin inhibitor (NU6140). We found that MV4-11-R is most sensitive to IDR E804 (an inhibitor of CDKs and the SRC-STAT3 pathway). The IC50 value of ABT-869 in MV4-11-R was decreased from 52 to 6.2 nM in the presence of 2 nM of IDR E804. Further validation of the therapeutic effect of IDR E804 in combination with ABT-869 in the MV4-11-R mouse xenograft model is ongoing. Targeting Survivin by shRNA and a dominant-negative vector (survivin-T34A) induced MV4-11-R to undergo apoptosis. Taken together, these results demonstrate that overactivation of STAT pathways and overexpression of survivin are the main mechanism of resistance to ABT-869; suggesting that the STAT pathways and survivin could be potential targets for the treatment of patients who develop resistance to FLT3 inhibitors.

Description: Abstract 2637 Background: Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTL) is a relatively rare but aggressive type of non-Hodgkins lymphoma that is more prevalent in Asia. The outcome of patients with disseminated stage is universally fatal. Progress in therapy has been slow and is based on combination of chemotherapy. MicroRNA are short non-coding RNA sequences that could regulate the expression of a large number of genes by inhibiting translation or leading to mRNA degradation. It has been implicated in tumorigenesis and has prognostic value across a wide range of malignancies including haematologic malignancies. We performed a comprehensive genome-wide miRNA expression profiling (MEP) of NKTL to identify deregulated miRNA and their potential role in NKTL biology. Method: MEP was performed using the Agilent human miRNA Microarray V2 (Agilent Technologies, Santa Clara, CA) on formalin fixed paraffin-embedded tissue (FFPE) (n=30) and NK cell lines (n=6) in comparison with normal NK cells. Differential expressed miRNA were identified using fold change and Significance Analysis of Microarray (SAM). Some of the differentially expressed miRNA were validated using quantitative polymerase chain reaction (q-PCR). The functional relevance of candidate miRNAs are assessed using miRNA mimics or inhibitors, and observing for apoptosis and growth arrest in the cell lines. Potential targets of candidate miRNAs are identified using predictive algorithms and significant negative correlation with gene expression data. The strongest candidate target genes are further verified using luciferase assay and q-PCR. miRNA and target gene relationship was further confirmed in the patients samples using immunohistochemistry for the protein expression on tissue microarray of NKTL. Results: Compared to normal NK cells, differentially expressed miRNAs in NKTL are predominantly downregulated. Re-expression of downregulated miRNAs, such as mir-101, mir-26a, mir26b, mir-28-5 and mir-363, reduced the growth of NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression are inversely correlated with the expression of deregulated miRNA in NKTL are significant enriched for genes involved in cell cycle-related, p53 and MAPK signaling pathways. We validated and confirmed the regulation of STMN1, and BLIMP1 by miR-101 and miR-30b respectively. In addition, miR-101, miR26a and miR-26b also affect the expression of BCL2 and IGF-1. We also performed immunohistochemical validation for selected target proteins and found over-expression of MUM1, BLIMP1 and STMN1 in NKTL, and notably, a corresponding increase in MYC expression. Conclusion: miRNA are dysregulated in NKTL. Since MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL as we have shown previously may contribute to the suppression of the miRNAs. These suppressed miRNA in turn lead to increase and aberrant expression of proteins and pathways of biological relevance to NKTL including cell cycle related genes, genes involved in p53 and MAPK signaling pathways as well as MUM1, BLIMP1 and STMN1. Reintroduction of these suppressed miRNA lead to death of NKTL cells and may be a potential therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

Description: It is well established that 17p13(del) and p53 mutations represent poor prognosis in MM patients. We have previously shown that p53 is a haploinsufficient tumor suppressor in myeloma and p53 expression level is correlated with functional status of the pathway. In addition, p53 expression levels do not always correlate with p53 genotype. The absence of p53 expression in myeloma cells severely abolishes the integrity of its downstream signaling response, thus, driving disease aggressiveness and drug resistance. There is a pressing need for a more effective treatment strategy for high-risk patients harbouring p53 abnormalities. PRIMA-1 is a first-in-class drug that was identified to specifically kill cancer cells harboring mutant p53. It has shown substantial efficacy in various solid tumors with mutant as well as non-mutant p53. However, the discovery of the anti-myeloma activity of PRIMA-1 is still at its infancy. By utilizing human myeloma cell lines (HMCLs) of different p53 status as our study model, we aim to identify the efficacy of PRIMA-1 in MM and to dissect the mechanisms behind PRIMA-1-induced toxicity. Despite its known ability to reactivate mutant-p53 function, we did not observe any restoration of mutant p53 transcriptional activities. The expression levels of the p53-direct-targets, p21, MDM2 and PUMA, were unchanged upon treatment in the p53-mutant-HMCLs. In fact, PRIMA-1 was found to be able to decrease the cell viability of all the HMCLs, irrespective of their p53 status (Mutant, WT, Null), strongly indicating a p53-independent response. Importantly, PRIMA-1 showed the highest cytotoxicity in a p53-deficient background (IC50 of 16uM in -/-, 55uM in WT/WT and 70uM in mutant HMCLs). This finding was validated when p53 silencing in a WT HMCL significantly enhanced its sensitivity to PRIMA-1. GEP analysis on the two most sensitive cell lines (JJN3 and KMS11) demonstrated significant enrichment of genes related to unfolded (p=1.90E-06 in JJN3 and 6.12E-08 in KMS11) and misfolded protein (p=2.99E-06 in JJN3 and 8.21E-08 in KMS11) responses. Subsequent validation by Western blot and qPCR confirmed the activation of ER stress pathway in response to PRIMA-1, evidenced by the up-regulation of ER stress markers such as GRP78, GADD34 and CHOP. Further investigations then revealed that the PERK/CHOP axis was most important as CHOP knockdown saw an attenuation of MM cells sensitivity to PRIMA-1-induced apoptosis. Recently, it was reported that cells with increased ER stress are more sensitive to bortezomib. Indeed, we showed that PRIMA-1 synergizes with bortezomib, and was able to rescue bortezomib resistance when both compounds were co-incubated in a bortezomib-resistant-HMCL. In conclusion, we identified a novel pathway by which PRIMA-1 exerts its toxicity in MM through induction of ER stress. This may be a novel therapeutic strategy to induce ER stress to sensitize cells to proteasome inhibitor. Owing to the versatility of the drug in targeting myeloma cells with or without p53 abnormalities; PRIMA-1 can potentially be used alone or in combination with other drugs such as bortezomib in a broad range of MM including those with high-risk disease. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a genetically complex disease that is becoming more common in today’s ageing population. Approximately 50% of MM harbour recurrent translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32. The translocation t(4;14)(p16;q32) is one of the most common translocation in MM and is associated with very poor prognosis. The multiple myeloma SET domain (MMSET), involved in the fusion to the IgH locus in t(4;14) MM, is universally overexpressed and has been suggested to play an important role in tumorigenicity in t(4;14) MM. In order to identify downstream functional targets of MMSET, we knocked down MMSET expression with shRNAs in KMS11, a t(4;14) MM cell line, and identified differentially expressed genes by gene expression microarray analysis. The results identified 321 down-regulated and 375 up-regulated genes upon MMSET knockdown. Of interest, the knockdown reduced the transcription levels of the transcription factor IRF4 (interferon regulatory factor 4), which has been shown recently to be required for myeloma cell survival. Quantitative PCR (QPCR) analysis confirmed the transcription levels of IRF4 were reduced upon MMSET knockdown in t(4;14) MM cells (KMS11, KMS18, KMS28BM). Western-blots analysis indicated MMSET knockdown reduced protein levels of IRF4, c-Myc and PARP, and IRF4 knockdown could also reduce protein levels of c-Myc and PARP. Flow cytometric analysis indicated silencing of MMSET or IRF4 could induce apoptosis in t(4;14) MM cells. QPCR and Western-blot analysis revealed that ectopic expression of MMSET could increase IRF4 expression in KMS11 cells. Using chromatin immunoprecipitation (ChIP), both MMSET and NFκB can bind to the promoter region of IRF4, and the binding region of MMSET is located 1 kb upstream of that of NFκB. Furthermore, endogenous NFκB p65 could be coprecipitated by anti-MMSET antibody, indicating the association of MMSET and NFκB. These data suggest that MMSET may be a coactivator for NFkB in the regulation of IRF4 expression. Luciferase reporter assays in KMS11 cells showed the activity of IRF4 promoters were decreased significantly upon MMSET knockdown, suggesting MMSET is an important functional element for IRF4 promoter. Cell proliferation assays and apoptosis analysis showed that silencing of MMSET could sensitize t(4;14) MM cells to Bortezomib and Melphalan killing. In myeloma xenograft, combined MMSET silencing with Melphalan or Bortezomib resulted in significantly greater inhibition of t(4;14) MM tumorigeness than treatments with either drugs alone. Importantly, we found both of Melphalan and Bortezomib could also reduce protein levels of MMSET and IRF4. QPCR analysis indicated that they inhibited MMSET and IRF4 expression through transcription control. These results might partly explain the additive mechanism of combination of MMSET knockdown and Melphalan or Bortezomib. Overall, our data indicated MMSET is involved in the regulatory network of IRF4 that is critical for t(4:14) MM cell survival, and its silence potentiates the effect of antimyeloma agents in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Description: Despite the proven efficacy of proteasome inhibitors like bortezomib (BTZ) in multiple myeloma, mantle cell lymphoma, and in an experimental setting in pediatric acute leukemia, development of drug resistance remains a primary hindrance. To further understand the molecular basis underlying this chemoresistance phenomenon, various leukemia cell line models with acquired resistance to BTZ were developed and characterized. One common characteristic was that acquisition of point mutations in PSMB5 and upregulation of the β5-subunit of the proteasome were key determinants of BTZ-resistance in vitro. However, it remains unclear how these drug resistance modalities translate to the overcoming of proteolytic stress imposed by proteasome inhibition. From this perspective, we here undertook a multi-modality (DNA, mRNA, miRNA) array-based analyses of human CCRF-CEM acute leukemia cells and two BTZ-resistant subclones [one with a low resistance level [(10-fold, CEM/BTZ7) and another subline with a high resistance level (140-fold, CEM/BTZ200)] to determine whether or not complementary mechanisms contribute to BTZ resistance. Gene expression profiling studies revealed markedly reduced proteolytic stress induction in drug resistant cells over a broad BTZ concentration range. Moreover, several genes involved in cytoskeleton regulation and vesicle migration were increased in resistant cells. Of all genes, myristoylated alanine-rich C-kinase substrate (MARCKS) was the most differentially overexpressed gene with 25- to 42-fold upregulation in CEM/BTZ7 and CEM/BTZ200 cells, respectively. These observations were corroborated at the protein level and solely included unphosphorylated MARCKS rather than phosphorylated MARCKS, which was marginally expressed in CEM/BTZ cells. Interestingly, MARCKS upregulation was also observed in other BTZ-resistant and leukemia cells (CEM and THP1) with acquired resistance to the proteasome inhibitor salinosporamide A and the immunoproteasome inhibitor PR924. Given the overexpression of MARCKS in proteasome inhibitor-resistant leukemia cells, we further explored whether or not MARCKS overexpression may serve as a predictive marker of BTZ resistance in clinical samples of acute leukemia patients. To this end, we examined primary patient specimens from a phase II childhood refractory/relapsed ALL trial in which BTZ is administered in two intensive re-induction regimens containing vincristine, prednisone, PEG-asparaginase, doxorubicin or cyclophosphamide and etoposide followed by methotrexate treatment. MARCKS expression was demonstrated in 64% of therapy-refractory pediatric leukemia specimens (n=44) wherein higher MARCKS expression trended (p=0.09) towards a dismal response to BTZ-containing chemotherapy. Finally, from a mechanistic perspective, we showed a concentration-dependent association of MARCKS protein with the emergence of ubiquitin-containing vesicles in the resistant cells. This association with MARCKS protein was lost upon exocytosis of these vesicles, which were found to be extruded and taken up in co-cultures with recipient HeLa cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance through vesicular exocytosis of ubiquitinated proteins in BTZ-resistant cells to overcome proteolytic stress over a broad range of cytotoxic BTZ concentrations. Disclosures Kaspers: Janssen-Cilag: Research Funding. Smeets:Novartis: Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cloos:Takeda: Honoraria.

Description: Recent studies have shown that 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, disrupts polycomb-repressive complex 2 (PRC2), and preferentially induces apoptosis in cancer cells, including acute myeloid leukemia (AML). However, the underlying molecular mechanisms are not well understood. The present study demonstrates that DZNep induces robust apoptosis in AML cell lines, primary cells, and targets CD34+CD38− leukemia stem cell (LSC)–enriched subpopulations. Using RNA interference (RNAi), gene expression profiling, and ChIP, we identified that TXNIP, a major redox control molecule, plays a crucial role in DZNep-induced apoptosis. We show that disruption of PRC2, either by DZNep treatment or EZH2 knockdown, reactivates TXNIP, inhibits thioredoxin activity, and increases reactive oxygen species (ROS), leading to apoptosis. Furthermore, we show that TXNIP is down-regulated in AML and is a direct target of PRC2-mediated gene silencing. Consistent with the ROS accumulation on DZNep treatment, we also see a signature of endoplasmic reticulum (ER) stress-regulated genes, commonly associated with cell survival, down-regulated by DZNep. Taken together, we uncover a novel molecular mechanism of DZNep-mediated apoptosis and propose that EZH2 may be a potential new target for epigenetic treatment in AML.

Description: Key Points JAK3-mediated phosphorylation of EZH2 resulted in EZH2 oncogenic function independent of its enzymatic activity. Targeted inhibition of JAK3 may be a promising treatment in NK/TL through suppressing noncanonical EZH2 activity.

Description: Abstract 3498 Nasal-type Natural Killer/T-cell lymphoma (NKTL) is an aggressive lymphoid malignancy associated with very poor survival. A better understanding of the molecular abnormalities underlying this disease will lead to a better therapy. We recently performed whole genome gene expression studies and identify a number genes that are differentially expressed in NKTL as well as pathways which are activated in NKTL. EZH2, one of the genes identified in our study to be aberrantly over-expressed in NKTL, is a H3K27-specific histone methyltransferase and a component of the polycomb repressive complex 2 (PRC2), which plays a key role in the epigenetic maintenance of repressive chromatin mark. To the best of our knowledge, the mechanism of EZH2 overexpression in NKTL has not yet been described. In this study, we showed that EZH2 overexpression in NKTL can be attributed to a deregulated MYC-miRNA-EZH2 regulatory axis where MYC activation represses miRNAs that normally downregulate EZH2. He functionally demonstrated this relationship using NKTL cell lines while also demonstrating the correlation between MYC activation and EZH2 expression in clinical samples through the analysis of gene expression data as well as histological detection of nuclear MYC and EZH2 protein using a tissue microarray containing 35 NKTL clinical samples using immunohistochemistry. We then investigated the functional role of EZH2 in NKTL. We showed that ectopic overexpression of EZH2 in both primary NK cells and NK cell lines led to a significant growth advantage. Conversely, knock-down of EZH2 in NK cell lines resulted in growth inhibition of tumor cells. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity was also able to confer growth advantage and rescue the growth inhibition upon endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene silencing activity. Indeed, EZH2 expression in clinical NKTL samples is associated with higher Ki67 staining implying a role in driving NKTL proliferation. We further demonstrated that EZH2 directly binds to the gene promoter of Cyclin D1 and EZH2 promotes the transcription of Cyclin D1 independent of its enzymatic activity. Consistent with its potential oncogenic role, depletion of EZH2 using an inhibitor called DZNep also induced significant growth inhibition in NKTL cells. Taken together, our study demonstrates an unconventional role of EZH2 in promoting oncogenic growth in NKTL and provides novel insights into the oncogenic function of EZH2 in human cancers. The pro-proliferative properties of EZH2 in NKTL support the rationale for using of EZH2 inhibitors in the treatment of NKTL. However, it is important to note that in some tumor, EZH2 may be mediating its oncogenic functions through non-enzymatic mechanism. This has critical implications on the choice of specific inhibitors of its enzymatic function or compounds that can deplete EZH2 as the most appropriate therapeutic approach. Since targeting of EZH2 is an active area of drug development at present, there is great potential for the development of better treatment modalities and this is especially important for aggressive cancers, such as NKTL, for which no effective curative treatment is currently available. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points This study has uncovered an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL. This study suggests that targeting EZH2 may have therapeutic usefulness in NKTL.

Description: Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation occurs in about 25% of acute myeloid leukemia (AML) patients and is associated with poor prognosis. Various FLT3 inhibitors of different chemical structure are under clinical investigation for the treatment of AML patients with FLT3 mutations. In contrast to their impressive potency in cell culture systems, FLT3 inhibitors as single agents in clinical trials only induce transient reduction of peripheral blasts, but not bone marrow blasts. Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. Small molecule HDAC inhibitors (HDACi) have proven to be a promising new class of anticancer drugs against hematological malignancies, as well as solid tumors. The HDACi, suberoylanilide hydroxamic acid (SAHA, Vorinostat) has been examined in a combinatory fashion with other classes of anticancer agents in acute leukemias, including cyclin-dependent kinase (CDK) inhibitors (flavopiridol), HSP 90 antagonists (17-allylamino- 17-demethoxygeldanamycin, 17-AAG), and estradiol analogs (2-methoxyestradiol, 2-ME). ABT-869, a multi-targeted receptor tyrosine kinase inhibitor, inhibits FLT3 phosphorylation and signaling and now is in active clinical development. In the current study, cell proliferation and apoptosis assays revealed that combining ABT-869 with SAHA led to synergistic killing of AML cells with FLT3 mutations in conventional cell culture and human stromal cell coculture models. To elucidate the molecular mechanism of the synergistic lethality between ABT-869 and SAHA, we compared the gene expression profiles of cells from FLT3 mutated leukemia cell lines, MV4-11 and MOLM-14, treated with DMSO control, ABT-869, SAHA and combination therapy using the Affymetrix microarray platform. Here, we focused on delineating a core gene signature unique to combination therapy and common to both MV4-11 and MOLM-14 cell lines, which could be crucial for the positive cooperation we observed. The core gene signature differentially induced more than two-fold by combination therapy in both cell lines included upregulation of FRY, LMO4, IFI16, ACADSB, and S100A8, and downregulation of PTP4A3 (PRL-3), ORC1L, MND1, ZNF85, DENND3, FAM119A, ACAT2, HMGCS1, DCS2, GSPG4, CBS, TUBE1. Overexpression of PRL-3, a metastasis-associated gene, has been found in different types of solid tumors and multiple myeloma. Modulation of PRL-3 expression level using genetic approaches demonstrated that PRL-3 played an essential role in the synergism in the therapeutic effect of ABT-869 and SAHA.