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Relapse after allogeneic hematopoietic stem cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusion of unselected donor lymphocytes (DLIs) enhances the graft-versus-leukemia (GVL) effect. However, because the infused lymphocytes are not selected for leukemia specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease (GVHD), related to the concurrent transfer of alloreactive lymphocytes. Thus, to minimize GVHD and maximize GVL, we selectively activated and expanded stem cell donor–derived T cells reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, and NY-ESO-1). Products that demonstrated leukemia antigen specificity were generated from 29 HCT donors. In contrast to DLIs, leukemia-specific T cells (mLSTs) selectively recognized and killed leukemia antigen–pulsed cells, with no activity against recipient's normal cells in vitro. We administered escalating doses of mLSTs (0.5 to 10 × 107 cells per square meter) to 25 trial enrollees, 17 with high risk of relapse and 8 with relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD seen. We observed antileukemia effects in vivo that translated into not-yet-reached median leukemia-free and overall survival at 1.9 years of follow-up and objective responses in the active disease cohort (1 complete response and 1 partial response). In summary, mLSTs are safe and promising for the prevention and treatment of AML/MDS after HCT. This trial is registered at www.clinicaltrials.com as #NCT02494167.

The promising activity of BET protein inhibitors (BETi’s) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm that develops from malignant B-lymphocytes in the outer edge or mantle zone of a lymph node. This is a sub-type of B-cell non-Hodgkin lymphoma characterized by rapid clinical progression and poor response rate to conventional chemotherapeutic drugs with recurrent relapse resulting in a short estimated 5-year overall survival (OS) of 2-5 years depending on the clinical risk. Combination therapies such as R-CHOP, R-DHAP, Hyper-CVAD, VcR-CAP constitute the front-line chemotherapeutic treatment landscape for MCL. Despite good initial response to the combination regimens, all patients develop resistance over time. The Bruton's tyrosine kinase inhibitor (BTKi) Ibrutinib and the proteasome inhibitor (PI) Bortezomib are FDA-approved therapies for refractory or relapsed (R/R) MCL with demonstrated high initial response rate in clinical trials. However, highly variable treatment response along with dose-limiting toxicities has limited the efficacy in real-world settings with the median progression-free survival (PFS) of Thus, the identification of novel drugs that function either alone or as combination to curb the oncogenic progression as well as to reduce drug-associated toxicities is of high clinical significance. We have designed a novel optimization-regularization-based computational prediction algorithm called "secDrug" that uses large-scale pharmacogenomics databases like the GDSC1000 to identify novel secondary drugs for the management of treatment-resistant B-cell malignancies. We hypothesize that combination of our predicted secDrugs with BTKi/ PI will be useful in curbing oncogenic progressions of R/R MCL and abrogate drug resistance through simultaneous inhibition of multiple oncogenic factors/pathways. When applied to BTKi/PI-resistant R/R MCL, the top predicted secondary drugs (secDrugs) were YM155 (Survivin inhibitor) and S63845 (selective MCL-1 inhibitor). Interestingly, both Survivin and MCL-1 are reported to be over-expressed in MCL, and their expression is strongly correlated with the oncogenic progression and survivability of the patients. To validate our in-silico predictions, we performed in vitro cytotoxicity assays with the top predicted secDrugs (YM155 and S63845) as single agents (IC50 for YM155 4.87±0.66 nM, for S63845 0.9±1.1 uM) as well as in combination with BTKi/PI against a panel of MCL cell lines representing PI/BTKi sensitive, innate resistant (representing refractory MCL) and clonally-derived acquired resistant (representing relapsed MCL). Our results showed that the YM155 and S63845 exhibited significant synergistic cell killing activities (Combination index/ CI value of 0.31±0.49 as calculated using Chou-Talalay's CI theorem, C.I>1 depicts synergism) alone and in combination with Bortezomib (PI) and Ibrutinib (BTKi), especially in R/R MCL cell lines. Further, our results also showed that both YM155 and S63845 in combination with BTKi/PI were able to significantly lower the effective dose of both BTKi/PI required to achieve desired therapeutic response by >12 times (Dose Reduction Index or DRI for YM155 in combination is 15.87±4.93; DRI for S63845 in combination is 12.34±2.67), thereby making the cell lines relatively more BTKi/PI sensitive. Next, we performed next-generation RNA sequencing analysis to identify mechanisms of secDrug action and synergy. Our Gene expression profiling and Ingenuity pathway analysis of the RNAseq data among YM155-treated MCL cell lines revealed eIF4-p70S6K signaling and mTOR signaling as the top canonical pathways. Our study thus identified YM155 and S63845 as potential novel candidates for repurposing as secondary drugs in combination with BTKi/PI for the treatment of R/R MCL. Currently, we are exploring the probable subclonal molecular mechanisms governing the synergistic drug action by using single-cell transcriptomics analysis. As both YM155 and S63845 have reported activity against cancer stem-ness, we will further investigate the effect of our novel drugs on the cancer stem-like cells in MCL, which have a potential role in treatment resistance. The secDrug algorithm promises to serve as a universal prototype for the discovery of novel drug combination regimens for treatment outcomes in any cancer type by enhancing sensitivity or overcoming resistance to standard of care drugs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

We have recently shown that XPO1 mutations are drivers of lymphomagenesis and occur across B-cell lymphomas, specifically in chronic lymphocytic leukemia (CLL), classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. The co-occurrence of other oncogenic events cooperating with XPO1 provides an opportunity for combined targeted therapy. Increased expression of the anti-apoptotic factor BCL2 has long been known to be a critical part of the pathophysiology of B-cell lymphomas. Recently, the oral BCL2 inhibitor, venetoclax, was approved in CLL and is currently being evaluated in clinical trials for other B-cell lymphomas. Selinexor is a potent, oral XPO1 inhibitor that was recently approved in multiple myeloma and diffuse large B-cell lymphoma. XPO1 inhibition exerts its antineoplastic effects by blocking key lymphomagenic pathways, such as NFκB, and decreasing the anti-apoptotic protein survivin. We therefore hypothesized that combining selinexor and venetoclax would have potential synergy and provide an oral precision combination therapy for relapsed, refractory lymphoma. We first set out to determine whether XPO1 mutant lymphoma cell lines showed differential response to either selinexor or venetoclax monotherapy. Five lymphoma cell lines; 3 diffuse large B-cell lymphoma (SU-DHL-6, SU-DHL-16, FARAGE) and 2 classical Hodgkin lymphoma (L428 and SUP-HD1), were subjected to next-generation sequencing (NGS) to assess for the presence or absence of XPO1 mutations. SUDHL-16 and SUP-HD1 were heterozygous for the XPO1 E571K hotspot mutation while SUDHL-6, FARAGE and SUP-HD1 were wildtype at the XPO1 locus. These 5 cell lines were used to assess sensitivity to Selinexor and/or Venetoclax. The CellTiter Glo assay was used to assess cell viability after 72 hours of treatment. Assays were performed in triplicate on 96-well plates that were read using a Spectramax plate reader. The XPO1 mutant cells showed increased sensitivity to selinexor (XPO1 mutant IC50 = 16-35nM; XPO1 WT IC50 = 41-231nM) as previously seen in conditional knockin mouse models of XPO1 mutant CLL (Figure A). Additionally, the XPO1 mutant cell lines showed increased sensitivity to single-agent venetoclax (XPO1 mutant IC50 = 2-13nM; XPO1 WT IC50 = 5-2853nM), an observation that has not previously been made (Figure B). Next, we tested the synergy of the combination of selinexor and venetoclax in the XPO1 mutant and wildtype cell lines. Increasing concentrations of the individual drugs were applied to each individual cell line in a 6x6 matrix. The cell viability percentage for each concentration was then entered into a synergy finder (www. synergyfinder.fimm.fi). The Bliss Independence model was used to calculate synergy of the Selinexor-Venetoclax combinations. As hypothesized, the combination of selinexor and venetoclax indeed showed synergy in both the wildtype and mutant XPO1 cell lines. Furthermore, the XPO1 mutant cell lines showed a higher degree of synergy compared to the wildtype cells (Figure C). Finally, a remarkable patient allowed us to test this combination ex vivo. This patient with CLL had undergone multiple therapies including chemoimmunotherapy, ibrutinib and venetoclax monotherapies. This patient had a founder XPO1 E571K mutation and also had acquired a BTK C481S ibrutinib resistance mutation and MYC amplification. These cells were unique in that they were easily able to be tested in ex-vivo culture to test sensitivity to different therapies. When tested with chemotherapy or ibrutinib they were completely resistant, and even with venetoclax they were fairly resistant; however, they remained sensitive to XPO1 inhibition with Selinexor. Selinexor and venetoclax showed remarkable synergy measured by a BLISS delta score of 18.78 (Figure D). In conclusion, inhibiting the nuclear exporter XPO1 and the anti-apoptotic factor BCL2 is synergistic in both XPO1 wildtype and mutant lymphoma. XPO1 mutant lymphomas show increased sensitivity to both selinexor and venetoclax. Additionally, selinexor and venetoclax showed a higher degree of synergism in XPO1 mutant lymphoma cell lines and were highly synergistic in primary XPO1 mutant CLL patient cells ex vivo. This combination is highly promising as an all oral alternative for relapsed, refractory lymphoma. Next steps include preclinical testing in mouse models in vivo using XPO1 mutant and wildtype mice crossed with mice overexpressing BCL2. Figure Disclosures No relevant conflicts of interest to declare.

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) that accounts for ~7% of all NHL in the U.S. MCL is associated with aberrant activation of the Wnt signaling pathway, which plays a key role in the survival and maintenance of MCL-initiating cells. Many MCL patients experience relapse and subsequent disease progression due to chemoresistance following initial therapy; hence, novel therapies are needed. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. The purpose of these studies was to examine the antitumor activity of SM09419 in preclinical models of MCL. SM09419 potently inhibited both CLK1-CLK4 (IC50 for all In vivo antitumor effects and tolerability of oral SM09419 (QD 20-21 days) were assessed in mice bearing REC-1 and JeKo-1 flank xenografts (n=5/group). In REC-1 xenografts, strong tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with 12.5, 25, and 50 mg/kg SM09419 (TGI 88% [p In summary, SM09419 potently inhibited SRSF6 phosphorylation Wnt signaling pathway activity, and cell proliferation and induced apoptosis in MCL cell lines. The strong in vivo antitumor effects observed as a single agent suggest that SM09419 may provide a clinical benefit for patients with treatment-resistant or refractory MCL. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

Introduction: Aberrant phosphorylation-mediated signaling is a well-established condition involved in the onset and progression of virtually all types of cancer. That not only depends on the over-activation of protein kinases, but also on the lack of the proper counterbalance of protein phosphatases, because of their decreased expression or activity. In turn, this is likely to be due to genetic and epigenetic alterations, or interactions with cellular inhibitors and inhibitory post-translational modifications, respectively. In Large Granular Lymphocyte Leukemia (LGLL), a rare lymphoproliferative disorder characterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, CLPD-NK), constitutive activation of phosphorylation-regulated survival pathways, especially the JAK2/STAT3 axis, but also PI3K/Akt and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since such pathways are modulated by the tyrosine phosphatase SHP-1 under normal conditions, it is conceivable that putative alterations of this phosphatase, and consequent inability to disrupt hyperactive pathways, play a role in LGLL pathogenesis. In this study, we assessed the ability of small molecules inducing the expression and stimulating the activity of SHP-1 to abrogate the survival pathways in LGL by counteracting aberrant signals generated by constitutively activated protein kinases. Methods: Peripheral blood specimens were collected from untreated patients with LGLL (both T-LGLL and CLPD-NK). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were further separated from PBMCs by using immunomagnetic beads (Miltenyi Biotec). LGLs were incubated either with acetyl-11-keto-β-boswellic acid (AKBA), a pentacyclic triterpene capable of inducing SHP-1 expression, or SC-78, a regorafenib-like derivative specifically stimulating SHP-1 activity, or both at increasing concentrations and discrete time points. After such treatments, LGLs underwent annexin V-PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the abundance of SHP-1 and the phosphatase activity thereof. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis. Results: Both AKBA and SC-78 proved effective at eliciting a significant level of apoptosis in the low micromolar range, exhibiting an additive effect when used in combination. Moreover, both compounds activated SHP-1 activity, as demonstrated by in-vitro phosphatase assays. As to the phosphorylation-dependent signals affected by SHP-1 activation, we observed a marked decrease in the phosphorylation status of STAT3, resulting in the downregulation of the STAT3-induced expression of downstream target genes (Mcl-1, survivin, Cyclin D and S1P5), as well as the dephosphorylation of Akt and p65, which confirmed the role of SHP-1 as a functional antagonist of survival pathways in LGLs. Conclusion: Taken together, our results support the newly emerging evidence that the targeted activation of SHP-1, which is already recognized as a tumor suppressor in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening new options for the treatment of LGLL. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Acute myeloid leukemia (AML) with TP53 mutation makes up ~13% of AML cases and is an aggressive, treatment-resistant subtype with dismal prognosis and limited therapeutic options. Aberrant activation of the Wnt signaling pathway is associated with AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells, making Wnt signaling inhibition a potential therapeutic modality for adverse AML. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway in a Wnt signaling reporter assay. The purpose of these studies was to examine the antitumor activity of SM09419 as a single agent and in combination with standard therapies in preclinical models of TP53 mutant (TP53mut) AML. In TF-1a and KG-1a AML cells with TP53 mutations, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt-related genes (LEF1, MYC, DVL2) and proteins vs. vehicle. The effect of SM09419 on cell proliferation was tested in 6 TP53mut AML cell lines. Proliferation was strongly impaired by SM09419 across all cell lines (EC50=0.23 + 0.056 µM). SM09419 significantly induced apoptosis in TF-1a and KG-1a cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In addition, SM09419 potently inhibited cell proliferation when tested in 27 leukapheresis-derived human primary AML cell lines (EC50=0.046 + 0.0061 µM) regardless of TP53 status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with cytarabine (Ara-C), venetoclax (VEN), or azacytidine (AZA) were assessed in mice bearing TP53mut flank xenografts (n=5-15/group). In TF-1a xenografts, SM09419 (12.5 and 25 mg/kg) induced significant tumor growth inhibition (TGI) vs. vehicle at D20 (TGI 55-56% [p In summary, SM09419 potently inhibited SRSF phosphorylation and Wnt pathway signaling and induced apoptosis in TP53mut AML cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of TP53 status. Strong in vivo antileukemic effects were observed with SM09419 as a single agent or in combination with other AML therapies, suggesting that it is a potential treatment for hard-to-treat AML subtypes such as TP53mut AML. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

Purpose: The combination of rituximab and chemotherapy has improved overall survival of diffuse large B-cell lymphoma (DLBCL) patients in recent decades. Relapsed/refractory DLBCL patients previously treated with rituximab-containing regimen had significantly poorer response to salvage therapy. To study the mechanisms of this resistance, our laboratory developed several rituximab resistant cell lines. Previous study from our group found demonstrated an aberrant imbalance in the levels of pro- and anti-apoptotic proteins in these cell lines, including Bak/Bak, Mcl-1/BCLxL/Survivin and inhibitor of apoptosis (IAP) family proteins resulting in acquired chemotherapy resistance. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets such as pRB, HIF-1, p73, NF-kB, and E2F1 as well as FOXO3a. It is also the major negative regulator of p53. Recently, we found that MX69 (a MDM2 inhibitor), which blocks the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, and induced apoptosis-dependent cell killing activity. Moreover, MX69 re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro. However, MX69 cannot be used clinically stressing the need to use a clinically available MDM2 inhibitor. To this end, we evaluated Idasanutlin, an investigational Nutlin family MDM2 antagonist, in B-cell lymphoma pre-clinical models. Methods: We used a panel of different DLBCL subtypes cell lines including activated B-cell lymphoma cell lines (TMD8, U2932); germinal center B-cell lymphoma (DOHH2, VAL, OCILY2, DH4, DH6, ROS50); rituximab-sensitive cell lines (Raji and RL); and rituximab/-resistant cell lines (Raji 4RH and RL 4RH). Cells were exposed to escalating doses of Idasanutlin (1nM-100µM) without or with other chemotherapeutic agents for 24, 48 and 72 hrs. Differences in cell viability were evaluated utilizing PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 10ng/ml for 30 minutes and followed by flow cytometric analysis. Western blot was used to detect the changes of MDM2, XIAP and PUMA expression before and after exposure to Idasanutlin 1uM for 24h. Results:In vitro exposure Idasanutlin to DLBCL cell lines demonstrated a dose- and time-dependent cell death. IC50 dosage of the cells was ranged from 0.7uM to 63.07uM at 48h. Low dose of Idasanutlin (1uM) was able to disrupt mitochondria and induced low mitochondrial membrane potential in both ABC and GCB cell lines. At molecular level, Idasanutlin reduced expression level of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP was reduced in Val and DHL4. Meanwhile, PUMA, the downstream of p53 activation, was increased after Idasanutlin exposure. Idasanutlin enhanced the anti-tumor activity of proteasome inhibitors (carfilzomib, ixazomib), Bcl-2 inhibitor venetoclax and Bryton kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed potent anti-tumor activity as a single agent in DLBCL pre-clinical setting. Idasanutlin was able to decrease cellular mitochondrial membrane potential. At molecular level, Idasanutlin decreased MDM2 and XIAP protein level and induced PUMA expression. Moreover, Idasanutlin enhanced anti-tumor activities of other small inhibitors. Taking together, Idasanutlin could be used as a novel and promising drug in the clinical setting of DLBCL with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

High expression of SKP2 (S-Phase Kinase Associated Protein 2) is associated with tumor cell growth and drug resistance in many solid tumors and hematological malignancies. However, the role of SKP2 in drug resistance/ relapse of multiple myeloma (MM) patients has not been fully investigated. To this end, we first analyzed two publicly accessible datasets (GSE6477, GSE9782) which demonstrated that relapsed MM patients showed higher expression of SKP2 compared to newly diagnosed patients (p=0.02). Furthermore, high expression of SKP2 was correlated with shorter overall survival compared to newly diagnosed MM patients with low SKP2 expression (p=0.0002). To investigate whether miRNAs could play a role in the dysregulation of SKP2 in MM, we exploited several miRNA-target prediction algorithms (Targetscan, miRDB, microT-CDS) to generate a selective miRNA library for subsequent screening. Over all, the screening results displayed 59 miRNA candidates predicated to target SKP2, 9 miRNAs were commonly identified by all three algorithms. Among these, miR-21-5p showed the highest potential binding score for SKP2 and was found to be in an inverse correlation with SKP2 in MM patient dataset (GSE16558). To address whether miR-21-5p/SKP2 axis may play a role in drug resistance in MM, we used two drug resistant MM cell lines (RPMI-8226R5 and MM.1R) to analyze the basal expression of SKP2 and miR-21-5p compared with that in their parental lines (RPMI-8226 and MM.1S, respectively). It turned out that the resistant MM cells displayed low expression of miR-21-5p and high expression of SKP2 whereas the parental lines showed the opposite pattern. Moreover, overexpression of miR-21-5p in both MM drug resistant cell lines significantly reduced both mRNA and protein levels of SKP2 further supporting that SKP2 could be a target of miR-21-5p. SKP2 mediates the ubiquitination of cyclin kinase inhibitor p27Kip1, a central negative regulator of cell cycle. In line with this, our immunoblot analysis confirmed that high SKP2 expressing drug resistant MM cells showed a low level of p27Kip1protein and low SKP2 expressing MM parental lines showed a high level of p27kip1 protein. To investigate the functional effects of miR-21-5p overexpression, we first performed FACS to determine if this overexpression would modulate cell cycle regulation of drug resistant MM cells. miR-21-5p overexpression increased in the proportion of cells at G1/G0 phase (RPMI-8226R5: 41.6 % to 59.1%, MM.1R: 19.5 % to 39.4%), decreased in the proportion of cells in S phase (RPMI-8226R5: 30.9 % to 25.5 %, MM.1R: 44.7 % to 32.2%) and G2/M phase (RPMI-8226R5: 15.5 % to 11.5 %, MM.1R: 27.6 % to22.4% ) compared with scrambled control. At the same time, immunoblot results showed that overexpression of miR-21-5p accumulated p27Kip1 and p21 (Cip1/Waf1), and suppressed CDK6 and Cyclin E cell cycle markers. Next, we examined the effect of miR-21-5p overexpression on cell proliferation and apoptosis in MM resistant cells using both MTT cytotoxicity assay and vital exclusion staining (trypan blue). In both assays, miR-21-5p mimics-transfected MM resistant cells showed significant reduction of cell viability and increase in apoptosis in a time-dependent manner compared with scrambled-transfected cells. Overexpression of miR-21-5p upregulated apoptotic markers: cleaved PARP-1, cleaved caspase-3, PUMA and BAX and down regulated anti-apoptotic markers: BCL-2 and survivin. Subsequently, efficacy of bortezomib (BTZ) was examined on miR-21-5p overexpressed MM resistant cells which were interestingly re-sensitized to BTZ in a dose- and time-dependent manner. In addition, our FACS experiment also confirmed that combination of BTZ and miR-21-5p mimics increased the percentage of annexin-V positive cells compared to scrambled and miR-21-5p mimics in the MM resistant cells. In conclusion, miR-21-5p is suppressed in MM resistant cells, which contributes to the drug resistance in MM through upregulation of SKP2. Overexpression of miR-21-5p induces apoptosis and cell cycle arrest in MM drug resistant cells by downregulating SKP2 gene expression and overcomes BTZ resistance in these cells. Our study provides a proof-of-concept that miR-21-5p could be utilized as a SKP2-targeting molecule which in combination with BTZ would be a promising therapeutic approach to overcome MM drug resistance. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for ~25% of all AMLs, carries a poor prognosis, and is prone to relapse despite targeted therapy. FLT3 mutations are associated with aberrant activation of the Wnt signaling pathway, which itself is implicated in AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. These studies examined the antitumor activity of SM09419 as a single agent and in combination with targeted and standard therapies in preclinical models of FLT3-ITD AML. In MV-4-11 and MOLM-13 AML cells carrying the FLT3-ITD mutation, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt pathway-related genes (CCND1, MYC, TCF7, DVL2). The effect on cell proliferation was tested in 8 AML cell lines with varying mutation profiles as well as 26 different leukapheresis-derived primary human AML cells. Proliferation was strongly impaired by SM09419 across all tested cell lines (average EC50=0.2 + 0.048 µM]); MV-4-11 and MOLM-13 cells had EC50 of 0.049 and 0.144 µM, respectively. SM09419 also potently inhibited proliferation in all primary AML cells (average EC50=0.048 + 0.0097 µM) regardless of FLT3 mutation status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). SM09419 also induced apoptosis in MV-4-11 and MOLM-13 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with either midostaurin (FLT3 inhibitor) or venetoclax (BCL2 inhibitor) and/or azacitidine were assessed in FLT3-ITD xenograft models (n=5-6/group). In MOLM-13 xenografts, SM09419 (12.5 and 25 mg/kg) induced strong tumor growth inhibition (TGI) vs. vehicle at Day 14 (TGI 52% [p In summary, SM09419 potently inhibited SRSF6 phosphorylation and Wnt signaling pathway activity and induced apoptosis in FLT3-ITD cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of FLT3 status. The strong in vivo antitumor effects observed as combination treatment suggest that SM09419 combined with standard therapies may provide a clinical benefit by slowing or preventing relapse in AML with a marker of poor prognosis such as FLT3-ITD. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. β2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.

Introduction: Multiple myeloma (MM) cells from patients with smoldering MM (SMM) and low-risk (LR) MM harbor genetic alterations typically seen in patients with high-risk (HR) disease. To test whether the bone marrow (BM) microenvironment plays a role in controlling growth of LR MM cells, we established an experimental model that mimics a HR microenvironment by co-culturing normal mesenchymal stem cells (MSCs) with HR MM cells. We previously have shown that MSC conditioned media (CM) promotes growth of MM cells more effectively than cell-cell contact, as adhesion to MSCs often promotes survival at the expense of proliferation. Therefore, we utilized CM and hypothesized that MSC CM is enriched with bioactive growth factors that facilitate proliferation of LR MM cells. The aim of the study was to test the effect of CM from unprimed and primed MSCs on the survival, growth, and molecular properties of LR MM cells, and identify molecular pathways that mediate these effects. Methods: Primed MSCs were prepared by co-culturing normal MSCs with BM-dependent MM lines for 5 days. MSCs were trypsinized, replated for 40 min followed by serial washing to remove MM cells. Molecularly classified CD138-selected LR MM cells from 8 newly diagnosed patients were treated with 50% primed CM or unprimed CM, or growth media (CONT) for 5 days. Growth and survival of primary MM cells was assessed by MTT assay and detection of annexin V/PI and KI67 by flow cytometry. Microarrays were performed on primed and unprimed MSCs (n=7) and on primary LR MM cells treated with primed and unprimed MSCs CM (n=3). Pathways were analyzed using Ingenuity. Ultra low depth WGS was performed to assess copy number variation. Protein arrays were performed to test levels of secreted factors in CM (n=7). Results: Growth of primary LR MM cells (n=8) was increased by primed CM 5.1±0.05 (p Primed MSC CM caused MM cell GEP70 score to increase resulting in change from LR to HR in 2 experiments and from an ultra LR score to an intermediate score in another. Pathway analyses on genes differentially expressed between primed CM- and unprimed CM-treated MM cells identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways. Top transcription regulators included FOXO3, TP53, E2F4, MYC and E2F1, whereas mir-16-5p and let-7 were the top microRNAs. Top significantly upregulated genes (>2 fold) by primed MSC CM included proliferation-related factors (MKI67, TOP2A, CCNB1, BIRC5 and RRM2), whereas underexpressed genes (< 2 fold) involved regulators of cell dormancy including BCL2 (survival), RICTOR (mTOR), HEY1 (NOTCH), JUN (AP-1) and CXCR4 (adhesion). Four genes we reported to powerfully predict progression of SMM to MM (Khan et al., Haematologica 2015) were highly upregulated in MM cells by primed MSC CM. WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes. IGF1 is a central MM growth factor and IGF binding proteins (IGFBPs) control its bioavailability. We recently reported that mesenchymal cells are the main source of IGFBPs in BM, with IGFBP2 being the most downregulated gene in MM bone (Schinke et al., CCR 2018). Expression and secretion of IGFBPs (particularly IGFBP2) by MSCs were significantly reduced by priming these cells with MM cells, whereas IGF1 levels remained unchanged. IGFBP2 markedly blocked IGF1-induced MM cell growth (p Conclusions: MSCs primed by HR MM cells mimic a HR microenvironment, reflected by reduced level of factors that restrain bioavailability of MM growth factors such as IGF1, resulting in shutdown of master regulators of cell dormancy, which then enable a MM cells to proliferate. Such a scenario is particularly applicable in SMM and LR disease where MM cells exhibit a low proliferative index and their expansion is accelerated in distinct HR BM microenvironmental niches such as focal lesions. Disclosures Epstein: University of Arkansas for Medical Sciences: Employment. Davies:Abbvie: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; TRM Oncology: Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.

Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34+38− AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk blasts and total CD34+ AML cells (P < .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Dendritic cell (DC) homeostasis, like all mature blood cells, is maintained via hierarchal generation from hematopoietic precursors; however, little is known about the regulatory mechanisms governing DC generation. Here, we show that prostaglandin E2 (PGE2) is required for optimal Flt3 ligand–mediated DC development and regulates expression of the Flt3 receptor on DC-committed progenitor cells. Inhibition of PGE2 biosynthesis reduces Flt3-mediated activation of STAT3 and expression of the antiapoptotic protein survivin, resulting in increased apoptosis of DC-committed progenitor cells. Reduced DC development caused by diminished PGE2 signaling is reversed by overexpression of Flt3 or survivin in DC progenitors and conversely is mimicked by STAT3 inhibition. PGE2 regulation of DC generation is specifically mediated through the EP1 and EP3 G protein PGE2 receptors. These studies define a novel DC progenitor regulatory pathway in which PGE2 signaling through EP1/EP3 receptors regulates Flt3 expression and downstream STAT3 activation and survivin expression, required for optimal DC progenitor survival and DC development in vivo.

Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.

There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive and fatal disease. We have examined 32 patients with smoldering, chronic, lymphoma and acute leukemia using Affymetrix HG-U133A2.0 arrays. Using the BRB array program, we identified genes differentially expressed in leukemia cells compared with normal lymphocytes. Several unique genes were identified that were overexpressed in leukemic cells, including TNFSF11, RGS13, MAFb, CSPG2, C/EBP-α, and TCF4; 200 of the most highly overexpressed ATL genes were analyzed by the Pathway Studio, version 4.0 program. ATL leukemia cells were characterized by an increase in genes linked to “central” genes CDC2/cyclin B1, SYK/LYN, proliferating cell nuclear antigen, and BIRC5. Because of its potential therapeutic importance, we focused our studies on the regulation and function of BIRC5, whose expression was increased in 13 of 14 leukemia samples. TCF4 reporter assays and transfection of DN-TCF4 demonstrated that TCF4 regulates BIRC5 gene expression. Functionally, transfection of ATL cells with BIRC5 shRNA decreased BIRC5 expression and cell viability 80%. Clinical treatment of ATL patients with Zenapax or bortezomib decreased BIRC5 expression and cell viability. These experiments represent the first direct experimental evidence that BIRC5 plays an important role in ATL cell viability and provides important insight into ATL genesis and potential targeted therapies.

To further investigate 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. Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3 ligand increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by short-hairpin RNA (shRNA) in MV4-11-R cells induces apoptosis and augments ABT-869–mediated cytotoxicity. Overexpression of survivin protects MV4-11 from apoptosis. Subtoxic dose of indirubin derivative (IDR) E804 resensitizes MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R xenograft model. Taken together, these results demonstrate that enhanced activation of STAT pathways and overexpression of survivin are important mechanisms of resistance to ABT-869, suggesting that the STAT pathways and survivin could be potential targets for reducing resistance developed in patients receiving FLT3 inhibitors.

The level of survivin was reported to be scarce in mouse megakaryocytes (MKs) compared with erythroid cells. Considering this finding and previously reported in vitro data showing decreased MK ploidy upon retroviral-mediated overexpression of survivin, we sought to examine whether ectopic survivin expression in the MK lineage might alter ploidy level in vivo. Here we report the generation of 2 tissue specific hematopoietic transgenic mouse models, one expressing survivin in both the erythroid and MK lineages and the other expressing survivin solely in the MK lineage. Survivin protein overexpression was confirmed in MKs and erythrocytes. Surprisingly, analysis of both transgenic mouse lines showed no detectable changes in MK number, ploidy level, and platelet and erythrocyte counts, as compared with control mice. We conclude that elevated survivin expression does not alter MK/erythroid lineage development and that elevated survivin, alone, does not interfere with MK ploidy in vivo.

Salinosporamide A (also called NPI-0052), recently identified from the marine bacterium Salinispora tropica, is a potent inhibitor of 20S proteasome and exhibits therapeutic potential against a wide variety of tumors through a poorly understood mechanism. Here we demonstrate that salinosporamide A potentiated the apoptosis induced by tumor necrosis factor alpha (TNF), bortezomib, and thalidomide, and this correlated with down-regulation of gene products that mediate cell proliferation (cyclin D1, cyclooxygenase-2 [COX-2], and c-Myc), cell survival (Bcl-2, Bcl-xL, cFLIP, TRAF1, IAP1, IAP2, and survivin), invasion (matrix metallopro-teinase-9 [MMP-9] and ICAM-1), and angiogenesis (vascular endothelial growth factor [VEGF]). Salinosporamide A also suppressed TNF-induced tumor cell invasion and receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis. We also found that it suppressed both constitutive and inducible NF-kappaB activation. Compared with bortezomib, MG-132, N-acetyl-leucyl-leucyl-norleucinal (ALLN), and lactacystin, salinosporamide A was found to be the most potent suppressor of NF-kappaB activation. Further studies showed that salinosporamide A inhibited TNF-induced inhibitory subunit of NF-kappaB alpha (IkappaBalpha) degradation, nuclear translocation of p65, and NF-kappaB-dependent reporter gene expression but had no effect on IkappaBalpha kinase activation, IkappaBalpha phosphorylation, or IkappaBalpha ubiquitination. Thus, overall, our results indicate that salinosporamide A enhances apoptosis, suppresses osteoclastogenesis, and inhibits invasion through suppression of the NF-kappaB pathway.

We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivinlox/lox mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

Patients with acute- or lymphoma-type adult T-cell leukemia (ATL) have a poor outcome because of the intrinsic drug resistance to chemotherapy. Protection from apoptosis is a common feature involved in multidrug-resistance of ATL. IAP (inhibitor of apoptosis) family proteins inhibit apoptosis induced by a variety of stimuli. In this study, we investigated the expression of IAP family members (survivin, cIAP1, cIAP2, and XIAP) in the primary leukemic cells from patients with ATL. We found that survivin was overexpressed in ATL, especially in acute-type ATL. Sodium arsenite was shown to down-regulate the expression of survivin at both the protein and RNA levels in a time- and dose-dependent manner, thus inhibiting cell growth, inducing apoptosis, and enhancing the caspase-3 activity in ATL cells. Nuclear factor-κB (NF-κB) enhances the transcriptional activity of survivin. Sodium arsenite suppressed the constitutive NF-κB activation by preventing the IκB-α degradation and the nuclear translocation of NF-κB. These findings suggest that survivin is an important antiapoptotic molecule that confers drug resistance on ATL cells. Sodium arsenite was shown to down-regulate the expression of survivin through the NF-κB pathway, thus inhibiting cell growth and promoting apoptosis of ATL cells.

The NKG2D receptor is a stimulatory receptor expressed on NK cells and activated CD8 T cells. We previously demonstrated that engaging the NKG2D receptor markedly improved the efficacy of a survivin-based DNA vaccine. The combination vaccine, encoding both the NKG2D ligand H60 and survivin, activates innate and adaptive antitumor immunity and results in better protection against tumors of different origin and NKG2D expression levels. Here we demonstrate that the enhanced vaccine efficacy is in part attributable to increased cross talk between lymphocytes. Depletion of CD8 T cells during priming reduces the vaccine-induced activation of dendritic cells (DCs) and NK cell activity. Depletion of NK cells during priming leads to reduced DC activation and CTL activity. However, depletion of CD4 T cells results in the activation of DCs, NK cells, and CD8 T cells and enhances NK cell activity. The pH60/Survivin vaccine also increases DCs and NK cells but decreases CD4 T cell homing to Peyer patches, presumably as a result of changes in the homing receptor profile. Thus, by preferentially activating and attracting positive regulators and reducing negative regulators in Peyer patches, this dual-function DNA vaccine induces a microenvironment more suitable for NK cell activation and T cell priming.

KBM5 cells, derived from a patient with blast crisis Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML), and imatinib-resistant KBM5 (KBM5-STI571) cells were found to express high levels of survivin. Inhibition of Bcr-Abl by imatinib significantly decreased survivin expression and cell viability in KBM5, but much less so in KBM5-STI571 cells. Inhibition of MEK, downstream of the Bcr-Abl signaling cascade decreased survivin expression and cell viability in both KBM5 and KBM5-STI571 cells. In addition, down-regulation of survivin by a survivin antisense oligonucleotide (Sur-AS-ODN) inhibited cell growth and induced maximal G2M block at 48 hours, whereas cell death was observed only at 72 hours in both KBM5 and KBM5-STI571 cells as shown by annexin V staining. Further, the combination of Sur-AS-ODN and imatinib induced more cell death in KBM5 cells than did either treatment alone. Down-regulating survivin also decreased colony-forming units (CFUs) in blast crisis CML patient samples. Our data therefore suggest that survivin is regulated by the Bcr-Abl/MAPK cascade in Ph+ CML. The facts that down-regulating survivin expression induced cell-growth arrest and subsequent cell death regardless of the cell response to imatinib and enhanced the sensitivity to imatinib suggest the potential therapeutic utility of this strategy in patients with CML, both imatinib sensitive and resistant.

Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-kappaB in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-kappaB. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammacnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-kappaB and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well.

Indole-3-carbinol, found in Brassica species vegetables (such as cabbage, cauliflower, and brussels spouts), exhibits antitumor effects through poorly defined mechanisms. Because several genes that regulate apoptosis, proliferation, and metastasis are regulated by nuclear factor-kappaB (NF-kappaB), we postulated that indole-3-carbinol must mediate its activity through NF-kappaB modulation. We demonstrated that indole-3-carbinol suppressed constitutive NF-kappaB activation and activation induced by tumor necrosis factor (TNF), interleukin-1beta (IL-1beta), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and cigarette smoke; the suppression was not cell type specific, because activation was inhibited in myeloid, leukemia, and epithelial cells. This activation correlated with the sequential suppression of the IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, p65 acetylation, and NF-kappaB-dependent reporter gene expression. The NF-kappaB-regulated gene products cyclin D1, cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), survivin, inhibitor-of-apoptosis protein-1 (IAP1), IAP2, X chromosome-linked IAP (XIAP), Bcl-2, Bfl-1/A1, TNF receptor-associated factor-1 (TRAF1), and Fas-associated death domain protein-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) were all down-regulated by indole-3-carbinol. This down-regulation led to the potentiation of apoptosis induced by cytokines and chemotherapeutic agents. Indole-3-carbinol suppressed constitutive NF-kappaB activation in mononuclear cells derived from bone marrow of acute myelogenous leukemia patients, and this correlated with inhibition of cell growth. Overall, our results indicated that indole-3-carbinol inhibits NF-kappaB and NF-kappaB-regulated gene expression and that this mechanism may provide the molecular basis for its ability to suppress tumorigenesis.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in almost all types of malignancies, making this protein a useful tool for the development of broadly applicable vaccination therapies. We used a recently identified HLA-A2 binding peptide and dendritic cells (DCs) from healthy donors to induce survivin-specific cytotoxic T lymphocytes (CTLs) in vitro. These T cells efficiently lysed target cells pulsed with the cognate peptide. Furthermore, survivin-specific CTLs recognized HLA-A2–matched tumor cell lines and primary malignant cells from patients with leukemia in an antigen-specific and HLA-restricted manner as demonstrated with the use of cold target inhibition assays and blocking antibodies. To validate the immunogenicity of survivin we performed the experiments in an autologous setting and used monocyte-derived DCs as targets. Interestingly, we found that DCs up-regulate survivin expression on stimulation with tumor necrosis factor α (TNF-α). However, these mature DCs were not recognized by survivin-specific CTLs, whereas they lysed autologous mature DCs pulsed with the antigenic peptide or transfected with whole tumor RNA purified from a survivin-expressing cell line. To further analyze the possible use of survivin-specific CTLs in cancer therapies, we induced survivin-specific CTLs using peripheral blood mononuclear cells (PBMNCs) and DCs from a patient with chronic lymphocytic leukemia (CLL). The in vitro–generated T cells efficiently recognized autologous malignant CLL cells, whereas they spared autologous-purified nonmalignant B cells or DCs. Our results demonstrate that survivin epitopes are presented on a broad variety of malignancies and can be applied in vaccination therapies.

Despite some exciting new leads in molecular pathogenesis, AIDS-defining primary effusion lymphoma (PEL) remains a fatal malignancy. The lack of substantial progress in the management of PEL demands innovative treatment approaches. Targeting intracellular molecules critical to cell survival is one unexplored strategy for treating PEL. Here we show that inhibition of signal transducer and activator of transcription–3 (STAT3) leads to apoptosis in PEL cells. STAT3 is constitutively phosphorylated in PEL cell lines BC-1, BCBL-1, and VG-1. Transduction of dominant-negative STAT3 and pharmacological STAT3 inhibition caused caspase-dependent cell death. Although STAT3 activation is known to induce expression of Bcl-2 family proteins, PEL cell apoptosis was independent of Bcl-2, Bcl-XL, or Mcl-1 protein expression. Instead, STAT3 inhibition induced transcriptional repression of survivin, a recently identified inhibitor of apoptosis. Forced overexpression of survivin rescued VG-1 cells from apoptosis induced by STAT3 inhibition. Our findings suggest that activated STAT3 signaling directly contributes to malignant progression of PEL by preventing apoptosis, acting through the prosurvival protein survivin. Since constitutive STAT3 activation and survivin expression have been widely documented in different types of cancers, their linkage may extend to many malignancies and be critical to their pathogenesis.

Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.

Second mitochondria-derived activator of caspases (Smac)/DIABLO is a mitochondrial protein that is released into the cytosol along with cytochrome c (cyt c) during the execution of the intrinsic pathway of apoptosis. Smac/DIABLO promotes apoptosis by neutralizing the inhibitory effect of the inhibitor of apoptosis (IAP) family of proteins on the processing and activities of the effector caspases. Present studies demonstrate that, upon engagement of the mitochondrial pathway of apoptosis, epothilone (Epo) B derivative BMS 247550, a novel nontaxane antimicrotubule agent, as well as the death ligand Apo-2L/TRAIL (tumor necrosis factor-α–related apoptosis-inducing ligand) induce the mitochondrial release and cytosolic accumulation of Smac/DIABLO, along with cyt c, in human acute leukemia Jurkat T cells. While it had no activity alone, ectopic overexpression of Smac/DIABLO or treatment with the N-terminus heptapeptide (Smac-7) or tetrapeptide (Smac-4) of Smac/DIABLO significantly increased Epo B– or Apo-2L/TRAIL–induced processing and PARP cleavage activity of caspase-3. This produced a significant increase in apoptosis of Jurkat cells (P

Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (BTK), has been FDA approved for Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, IBR responses are incomplete in most cases, and of short duration for MCL and higher-risk CLL subtypes. We hypothesize that intrinsic resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be co-targeted with BTK inhibition to achieve deeper and more durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generated synergistic cytotoxicity with IBR in MCL lines as well as circulating leukemic B cells in 13/19 CLL and 4/5 MCL patient samples treated ex vivo (Jayappa KD et al. ASH 2015; Portell CA et al. ASH 2014). However, the sensitivity to VEN or IBR+VEN was highly variable among patient samples, and did not correlate with the diagnostic genetic lesions in the CLL/MCL cells or clinical characteristics of the patients. Here, we show that resistance to IBR+VEN can be induced by interaction with soluble factors found in the in vivo "macroenvironment" of the circulating cancer cells. To gain insight into changes in signaling pathways that might underlie mechanisms of drug synergy and resistance, we analyzed drug resistant MCL lines treated with IBR, VEN, and the combination by Reverse Phase Protein Arrays. We observed downregulation of PI3K-Akt, MAPK, JAK-STAT, and NOTCH signaling after IBR and IBR+VEN treatment, cleavage of caspases and PARP after VEN-treatment, and greatly enhanced cleavage of caspases and PARP after IBR+VEN treatment. A notable exception was significantly increased phosphorylation on p65 S536 of the NF-kB pathway at longer times after IBR+VEN treatment, indicating a possible role of NF-kB signalling in generating resistance to IBR and VEN in cells that survived treatment. To determine whether extrinsic factors in the cancer cell environment might be able to induce a drug-resistant phenotype, we co-cultured or pre-incubated PBMC with a diverse panel of stromal cells, cytokines, and other agonists. We found that the combination of soluble CD40L, IL-10, and CpG DNA (agonist mix) generated nearly complete resistance to IBR+VEN in CLL and MCL patient samples, with CpG DNA being the most effective single agent. Every sample treated with agonist mix displayed increased resistance to IBR+VEN drug combination, suggesting that responsiveness transcends genetic heterogeneity and reflects the developmental lineage of the cancer cells. We investigated whether the extrinsic agonists induce drug resistance by activating the NF-κB pathway, by analyzing nuclear localization of NF-kB transcription factors RelA and RelB using ImageStream and cell fractionation. We observed robust activation of alternative-NF-kB signaling in primary CLL and MCL cells treated with agonist mix, with little effect on canonical NF-κB. PKC, MAPK and PI3K-Akt signaling also showed evidence of activation by agonist mix. Agonist mix treatment also caused significant overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, but not Bcl-2. Inhibitors of NF-κB blocked RelB translocation and overexpression of Mcl-1, Bcl-xL and survivin. Inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame drug resistance induced by agonist mix. Inhibitors of the other activated pathways (MAPK, PI3K-Akt, PKC) did not block agonist-induced drug resistance at pharmacologically relevant concentrations. To determine whether extra-nodal agonists in the blood of patients could generate resistance to IBR and VEN, we analyzed drug cytotoxicity in CLL patient PBMCs cultured with autologous plasma. We found that autologous plasma was able to induce increased resistance to IBR+VEN in 3/7 CLL samples and this resistance was blocked by treatment with an NF-kB inhibitor. In conclusion, soluble agonists in the patient macroenvironment of circulating CLL/MCL cells can generate resistance to IBR+VEN by activating alternative-NF-kB signaling and over-expression of multiple anti-apoptotic proteins. Inhibitors of NF-kB or of the upregulated anti-apoptotic proteins overcame IBR+VEN resistance generated by these extrinsic factors. We suggest that survival signals generated by extra-nodal agonists in the patient macroenvironment generate drug resistance in CLL and MCL, and that improved responses could occur with interventions that block these survival signals. Disclosures Portell: AbbVie: Research Funding; Roche/Genentech: Research Funding; Infinity: Research Funding; Acerta: Research Funding. Wulfkuhle:Theranostics Health, LLC: Equity Ownership. Petricoin:Perthera, Inc.: Consultancy, Equity Ownership; Ceres Nanosciences, Inc.: Consultancy, Equity Ownership, Patents & Royalties; Avant Diagnostics, Inc.: Equity Ownership. Williams:University of Virginia: Employment; Janssen and Pharmacyclics: Research Funding.

Introduction: It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promotes lymphomagenesis. Over-expression of the type II protein arginine methyltransferase (PRMT5) enzyme has been associated with increased cell proliferation and survival in both solid and blood cancers. We have reported that PRMT5 is essential for EBV-driven B cell transformation and that it regulates the EZH2, EED and SUZ12, components of the Polycomb-repressive complex 2 (PRC2) complex via transcriptional silencing of RBL2and hyper-phosphorylation of RB1 in aggressive lymphomas. Here we report a mechanistic assessment of how PRMT5 over-expression supports the WNT/β-CATENIN pathway at multiple levels and drives oncogenic β-CATENIN target genes in lymphoma. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells was achieved by infecting with sh-PRMT5 lentivirus (or sh-GFP control), a selective small molecule PRMT5 inhibitor (Alinari et al, Blood 2015, CMP5) or CRISPR/CAS9 PRMT5 deletion. Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by MTS proliferation assay and FACS analysis. WIF1 protein detection in cell culture media was performed by ELISA. Results: PRMT5 regulated WNT/β-CATENIN signaling by direct transcriptional repression of AXIN2 and WIF1, two proteins that negatively regulate this pathway. PRMT5 inhibition with shRNA, CRISPR/CAS9 deletion, or CMP5 led to restored expression of AXIN2 and WIF1 transcript and protein that was associated with transcriptional repression of WNT/β-CATENIN target genes CYCLIN D1, MYC, and SURVIVIN. With PRMT5 inhibition, we observed differential enhanced recruitment of co-repressors LSD and HDAC2 and loss of associated epigenetic marks H3K4Me3 and H3K9Me (associated with the LSD demethylase) and H3K9Ac and H3K14Ac (associated with HDAC2) at each b-CATENIN target promoter. PRMT5 inhibition was found to reduce recruitment of co-activators CBP and MLL1 and respective epigenetic mark H3K4me3. The de-repression of AXIN2 and WIF1 was associated with loss of phospho-AKT (S450, S473) and down-stream survival pathways. Reduction in phospho-AKT was attributed to physical association between AXIN2 and the down-stream activity of secreted WIF1 in media of lymphoma cells treated with PRMT5 inhibition or CRISPR/CAS9 PRMT5 deletion. Furthermore, reduction of phospho-AKT prevented dimerization of MLL1 leading to dissociation of the BCL9-Pygopus TCF1/b-CATENIN transcriptional activation complex at MYC, CYCLIND1, and SURVIVIN promoters. Our observations show that PRMT5 is an important epigenetic regulator that governs the expression WNT/β-CATENIN-driven oncogenes c-MYC, CYCLIND D1 and SURVIVIN. PRMT5 inhibition restores regulation at several levels that converge on AKT signaling; (i) AXIN2-AKT interaction and (ii) WIF1 inhibition of WNT signaling. The restored regulation occurs via modulation of the downstream transcriptional machinery that is supported by constitutive AKT activity. This data identifies a novel pathway to interfere with WNT/β-CATENIN signaling and validates the driver role orchestrated by PRMT5 in lymphoma. Disclosures Baiocchi: Essanex: Research Funding.

Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient. Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice. Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib). Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. Disclosures Konopleva: Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it. Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis. Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. Disclosures No relevant conflicts of interest to declare.

Leukemia stem-like KG-1a cells escape the synergistic cytotoxic effect of arsenic trioxide and aclacinomycin: regulated by survivin Yongbin YE1 , Xiaojun.XU1 , Liu Qifa2 Correspondence to: Xiaojun XU. E-mail: doctorxu@163.com 1Department of Hematology, Zhongshan Hospital of Sun Yat-sen University & Zhongshan City People Hospital, Zongshan 528403 2Department of Hematology, Nanfang Hospital of Southern Medical University, Guangzhou,510515 Abstract AIM OF STUDY: Arsenic trioxide combined with aclacinomycin has a synergistic cytotoxic effect on leukemia stem cell-like cells KG-1a in our pervious study, however, there are still a part of KG-1a cells escaped the synergistic cytotoxicity, survivin may plays in a key role in this process. In this study, we have studied the interaction and mechanism of survivin in regulating leukemia stem-like KG-1a cell escape the synergistic cytotoxic effect of arsenic trioxide and aclacinomycin. MATERIALS AND METHODS: The anti-proliferation effect was detected by CCK-8 and colony-forming assay, protein-protein chip assay was used to analysis the expression level of survivin before and after combination treatment. The induction of apoptosis and cell cycle arrest in KG-1a cell line were detected by FACS, the expression of related signal pathway protein was detected by western blot. RESULTS: The anti-proliferation of KG-1a cells caused by arsenic trioxide or aclacinomycin showed a time- and dose-independent manner. However, protein-protein chip assay showed that the expression of survivin had a significant increase after the combination treatment (p Key words: arsenic trioxide,aclacinomycin, Acute leukemia; survivin; chemotherapy resistant Disclosures No relevant conflicts of interest to declare.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murine survivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin(140), similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin(121) that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin(40), lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin(140) and survivin(121) inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin(140) mRNA, whereas survivin(121)-specific transcripts were detected in all tissues, while those representing survivin(40) were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis. (Blood. 2000;95:1435-1442)

Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin. Disclosures No relevant conflicts of interest to declare.

Chemotherapy is not curative for the majority of patients with acute myeloid leukemia (AML) due to the presence of leukemia stem cells (LSCs) and the emergence of other clonal populations that exhibit resistance to cytotoxic therapy. Understanding the pathways responsible for the development of chemotherapy resistance is critical for developing novel strategies that more effectively target AML. We have previously demonstrated that the MUC1 oncogene is expressed on AML cells including LSCs. MUC1 is a heterodimeric glycoprotein where the MUC1-C subunit functions as an oncoprotein. Importantly in AML, MUC1-C facilitates the nuclear translocation of active β-catenin necessary for downstream effectors including survivin, a negative regulator of apoptosis. Recent data has demonstrated that survivin inhibition is crucial in conferring susceptibility to chemotherapeutic agents in a leukemia model. In the current study, we sought to examine the effect of MUC1-C inhibition on survivin levels and the sensitivity of leukemic cells to cytotoxic chemotherapy. To assess the effect of MUC1-mediated signaling on survivin expression, MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C in two AML cell lines, MOLM-14 and THP-1. Silencing of MUC1-C was confirmed by flow cytometric and western blot analyses and resulted in the downregulation of β-catenin and its target, survivin, at both the protein and mRNA level. In contrast, MUC1-C overexpression led to increased survivin expression. The role of MUC1-C as a mediator of resistance to cytotoxic chemotherapy was assessed. A stable MUC1-C gene knockdown of the AML cell line, MOLM-14, was generated using CRISPR/Cas9 technology. The MOLM-14 CRISPR and MOLM-14 wild-type (WT) cell lines were independently treated with increasing doses of the cytotoxic chemotherapeutic agent, cytarabine (Ara-C 50-1000 nM). The MOLM-14 CRISPR cell line demonstrated reduced cell viability utilizing an ATP-based luminescence assay (CTG, Promega) as compared to the MOLM-14 WT cell line at 72 hours (14% vs. 32%) and 96 hours (6% vs. 28%) after treatment with Ara-C. The results demonstrate that MUC1-C confers resistance to chemotherapy, and that the loss of MUC1-C in leukemic blasts significantly increases AML susceptibility to cytotoxic chemotherapy. Next, we investigated if the functional inhibition of MUC1-C would increase the sensitivity of AML to Ara-C. A novel cell-penetrating peptide, GO-2O3, binds to the MUC1-C subunit and blocks MUC1-C homodimerization and function. Two AML cell lines, MOLM-14 and MV4-11, were treated with increasing doses of Ara-C (25-1000nM) and GO-2O3 (1-5uM) to establish dose-dependent cytotoxicity curves. Based on the cytotoxicity curves, doses of Ara-C (50, 100, 125 nM) and GO-2O3 (1.0, 1.5, 2.0 uM) were selected for combination therapy. Analysis at 48 hours utilizing CTG demonstrated statistically significant synergy validated by the combination index (CI) calculated through CompuSyn [MV4-11 (0.54) and MOLM-14 (0.86)] where CI values < 0.90 indicate drug synergy. These results were confirmed in both the MOLM-14 and MV4-11 cell lines by staining for Annexin V/PI with FACS analysis after 48 hours of treatment. In MOLM-14, apoptosis and necrosis were noted as follows: no treatment (1.5%, 0.6%), GO-2O3 (3.7%, 6%), Ara-C (12%, 9.8%), and the combination (17%, 34%). In MV4-11, apoptosis and necrosis were noted as follows: no treatment (2.1%, 0.9%), GO-2O3 (4%, 6%), Ara-C (17.3%, 11.2%), and the combination (14%, 40%). The functional inhibition of MUC1-C in combination with Ara-C resulted in both decreased cell viability and increased cell death as compared to either agent alone. In conclusion, the data demonstrates that MUC1 expression on AML cells plays a critical role in conferring resistance to chemotherapy. Via its effector, survivin, MUC1-C inhibition renders leukemia cells more susceptible to cytotoxic injury in synergy with Ara-C. A clinical trial evaluating the combination of Ara-C and GO-203 in patients with relapsed AML is planned. Disclosures Kufe: Genus Oncology: Consultancy, Equity Ownership.

INTRODUCTION: Novel therapies are needed for acute myeloid leukemia as only ~60% of children are cured despite maximally intensive cytotoxic chemotherapy. Functionalized gold nanoparticles (AuNP) are utilized for many biomedical applications and represent a potentially novel therapeutic approach in leukemia. We enhanced this technology by developing an AuNP system that selectively releases molecularly targeted drugs in leukemia cells. METHODS: AuNPs were functionalized with short, double-stranded oligonucleotides with sequence complementarity to genes overexpressed in or unique to a leukemia cell (e.g. survivin or AML/ETO). Only the anti-sense oligonucleotide is covalently bound to the nanoparticle via a thiol linker. Thus, after entering leukemia cells, the endogenous targeted oncogene mRNA can bind to its complementary sequence on the AuNP and displace the non-covalently bound oligonucleotide which, in our system, is conjugated to the multi-tyrosine kinase inhibitor dasatinib. As a binary reaction, the amount of dasatinib-conjugated oligonucleotide released from the nanoparticle is directly proportional to both the presence and abundance of the complementary mRNA present in a cell. We evaluated AuNP uptake into multiple AML cell lines, as well as normal hematopoietic cells. The effect of dasatinib-AuNPs on dasatinib-sensitive leukemia cell lines was evaluated using proliferation assays, annexin V staining, and cell colony assays. Toxicity in T-cells and CD34+ cells was assessed with T-cell activation and p-SRC assays, respectively. RESULTS: Conjugation of dasatinib to an oligonucleotide complementary to a region of the survivin gene did not perturb its ability to inhibit SRC and c-KIT kinases in vitro. Leukemia cells demonstrate highly efficient AuNP uptake when cultured alone or with up to a 100-1000 fold excess of normal bone marrow cells. Treatment of K562 leukemia cells, containing a BCR/ABL translocation and high levels of survivin mRNA, with dasatinib-AuNPs resulted in dose-dependent p-SRC and p-CRKL inhibition. Furthermore, K562 cells also showed significantly impaired proliferation, increased apoptosis, and formed fewer colonies in methylcellulose. Conversely, normal T-cells and CD34+ cells, which express less survivin than leukemia cells, were significantly less affected by dasatinib-AuNPs than dasatinib alone as measured by T-cell activation assays and p-SRC levels, respectively. CONCLUSIONS: This method of using functionalized AuNPs to deliver and activate dasatinib in leukemia cells augments drug efficacy while minimizing toxicity and thus represents a novel therapeutic strategy for AML. Ongoing work is underway to characterize the mechanism of preferential AuNP uptake in leukemia cells and assessing in vivo activity using a murine xenotransplantation model of leukemia. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

In diffuse large B-cell lymphoma (DLBCL) the addition of rituximab to front line multi-agent therapy has changed the biology and clinical outcome of patients with relapsed/refractory disease. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines, which also display significant resistance to a wide range of chemotherapy agents. These therapy resistant cell lines (TRCL)s have multiple deregulations the apoptotic response pathway, including a loss of Bak and Bax. We previously demonstrated that small molecule mimetics of the second mitochondrial activator of caspases (SMAC), which antagonizes the inhibitor of apoptosis (IAP) family of proteins, could boost response to chemotherapy agents and extend survival in TRCL xenograft animal models. To investigate how SMAC mimetics overcome chemotherapy resistance in TRCL (RL-4RH and Raji-4RH) models we used RNA interference to transiently knockdown expression of individual IAPs (cIAP1, cIAP2, livin, survivin, and XIAP), and then exposed those cells to a panel of chemotherapy agents. Apoptosis induction was analyzed by flow cytometry staining for annexin V and Sytox blue (a DNA stain). While SMAC mimetics can decrease levels of the cellular inhibitor of apoptosis proteins (cIAP1/cIAP2), siRNA knockdown of cIAP1/cIAP2 did not increase chemotherapy-induced apoptosis. On the other hand, siRNA knockdown of XIAP in TRCLs substantially increased rates of apoptosis compared to cells transfected with non-targeting siRNA (5% in controls vs. 22% in XIAP knockdown RL-4RH cells). An additional 24% of XIAP knockdown RL-4RH cells showed signs of necrosis compared to 0.5% in control RL-4RH. Results were confirmed by western blot (PARP cleavage). In addition, siRNA XIAP knockdown re-sensitized TRCLs (Raji-4RH and RL-4H) to the cytotoxic effects of multiple chemotherapy agents (gemcitabine, etoposide, and vincristine). To investigate if XIAP knockdown could improve chemotherapy response in vivo we created a TRCL (Raji-4RH) that stably expresses XIAP targeting shRNA along with luciferase to enable in vivo imaging. SCID mice were inoculated with either the Raji-4RH XIAP knockdown or Raji-4RH non-targeting shRNA control cell line (10x106 cells per animal given i.v.). Tumors were allowed to engraft for 7 days prior to treatment administration. Animal were either given no treatment, or rituximab in combination with ifosfamide, carboplatin, etoposide (RICE) and mesna. Following treatment animals were imaged on a weekly basis to track tumor progression. At the time of the submission disease progression was present in animals in the observation groups, and in animals inoculated with non-targeting control shRNA Raji-4RH treated with RICE. InterestingHowever, animals inoculated with XIAP knockdown Raji-4RH and treated with RICE therapy remain tumor free with no detectable disease. In summary, XIAP acts as a key cell survival regulator in rituximab-chemotherapy resistant lymphoma models. Downregulation of XIAP can enhance chemotherapy activity and promote tumor cell death. Additionally, XIAP knockdown appears to enhance the activity of RICE, a commonly used regiment in the relapsed/refractory setting for DLBCL patients. Our results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against relapsed/refractory B-cell lymphoma. Disclosures Czuczman: Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board; MorphoSys: Consultancy; Celgene: Employment.

Multiple sequential genetic and epigenetic alterations, including alterations in microRNAs (miRNAs), underlie the development and progression of cancers. For example, overcoming cellular senescence is an early step in cancer pathogenesis. We show here that a tumor-suppressive miRNA, microRNA-16/miR-16 has a potential to induce cellular senescence in cutaneous T-cell lymphoma (CTCL). Firstly, to examine the role of miR-16 in the progression of primary CTCL, we performed q-PCR analysis against early (n=26) and advanced (n=14) Mycosis Fungoides samples. As for control (CD4+ T-cells), we used samples from patients with atopic dermatitis (AD; n=18). We found that expression of miR-16 in both early and advanced stages were significantly lower than AD, with the expression of advanced showing significantly more reduced than early stage. These results suggest that miR-16 is associated with lymphomagenesis from early to advanced stages of CTCL. To examine in vivo tumor-suppressive effects of miR-16, we used NOD/Shi-scid IL2Rgnull (NOG) mouse xenograft model, which would die around day 30-40 after CTCL cells (My-La, MJ, HH or HUT78) injection by invasion and metastasis (Ito et al, Blood 2014). By use of the xenograft model, we found that miR-16 transduced CTCL cells transplanted NOG mice showed significant prolonged survivals than control xenograft mice. We previously demonstrated that miR-16 did not inhibit migration capability of CTCL cells. When we considered that miR-16 successfully contribute to extend survival of mice models despite of lacking migration-inhibition capability, the result suggested that other tumor suppressive mechanisms might exist. Therefore we conducted functional analysis of miR-16, and found that miR-16 could induce cellular senescence against CTCL cells. In CTCL cells that expressed wild-type p53 (My-La and MJ), forced expression of miR-16 enhanced p21, which is a key mediator of senescence, in a p53 dependent manner. Because miR-16 family members are also able to directly suppress the polycomb group protein Bmi1, which is known to negatively regulate p21, we assessed Bmi1 expression in miR-16-transduced CTCL cells and found it to be diminished in all four CTCL cell lines tested. When we knocked down BMI1, expression of p21 was elevated in cells expressing wild-type p53 and the incidence of senescence was increased. We further demonstrated that Bmi1 could directly interact with the promoter lesion of CDKN1A/p21 by cross-linking/chromatin immunoprecipitation assays. From these results, miR-16 has potential to induce cellular senescence via targeting Bmi1-p21 pathway in p53-wild type CTCL cells. Although miR-16 could not induce senescence in p53 non-functional CTCL cells (HH and HUT78), in that case, we found that miR-16 can compensatory induce apoptosis by negative regulation of Bmi1-Survivin pathway. To elucidate this senescence-apoptosis switch mechanism, we examined co-transfection of miR-16 and siCDKN1A/p21 against p53 wild-type CTCL cells. We found that co-transfection of miR-16 and siCDKN1A significantly decreased senescent cells and increased apoptotic cells, with downregulation of Bmi1 and Survivin. In addition, miR-16 negatively regulated cyclinD1-Rb pathway regardless of p53 status, leading to cell growth inhibition against CTCL cells. Together, these results strongly support our idea that presence or absence of functional p53-p21 is an essential factor in miR-16-mediated senescence-to-apoptosis switch in CTCL. Finally, because a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA, vorinostat) has been recently applied and marks good responses in advanced CTCL, we tried to examine whether SAHA might share essential targets with miR-16 in CTCL lymphomagenesis. We found that SAHA restored miR-16 and its essential targets (p21, Bmi1, Survivin), and induced senescence in CTCL cells with wild-type p53 and apoptosis in cells with non-functional p53. Moreover, we found that T or NK/T-cell lymphoma cells (n=8) derived from various subtypes also showed similar tumor-suppressive effects by miR-16 transduction or SAHA treatment. In conclusion, through detailed study of miR-16 function, we have shed light on the mechanism by which lymphoma cells avoid senescence. Furthermore, identification of essential SAHA targets may provide supporting evidence of its clinical application for various non-Hodgkin T/NK-cell lymphomas. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: SINE compounds are a family of small-molecule drugs that inhibit XPO1 mediated nuclear export, resulting in nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IκB and subsequently in specific cancer cell death. Selinexor is a clinical stage SINE compound currently in human phase I/II clinical trials in patients with solid and hematological malignancies. Glucocorticoid Receptor (GR) is an XPO1 cargo and dexamethasone (Dex) acts as a GR agonist and inhibits NFκB activity. The combination of selinexor with Dex (Sel-Dex) shows enhanced anti-tumor potency in vitro and in vivo. The current study aimed at deciphering the molecular changes that contribute to the synergism of Sel-Dex. Methods: Total RNA and whole protein cell lysates from MM cell lines treated with selinexor with or without dexamethasone were analyzed by quantitative PCR and by immunoblots. Localization of GR was evaluated by immunofluorescence microscopy. GR and NFκB transcriptional activity was analyzed using ELISA assays (Affimetrix and Thermo Scientific). RNA from naïve and drug treated cells was analyzed by deep sequencing (by Asuragen). Results: Dexamethasone, but not selinexor, induced phosphorylation of GR. Selinexor blocked nuclear export of phosphorylated GR, enhancing GR transcriptional activation. Deep sequencing revealed a set of genes, whose level of expression was synergistically modified by Sel-Dex. Those genes belong to several pathways including GPCR signaling, cell metabolism and ERK, TGF-β and PI3-AKT signaling. Among the genes that were synergistically up regulated by the combination were genes from the Early Growth Response family (EGR1, EGR3, EGR4). EGR1 is a tumor suppressor protein which down-regulates survivin and triggers Caspase signaling and cell death. Interestingly, EGR1 also mediates the cytotoxic effects of bortezomib and lenalidomide in multiple myeloma cells. The Glucocorticoid-Induced Leucine Zipper (GILZ) gene was also up regulated by Sel-Dex combination. It has been shown by others that silencing GILZ expression decreased the therapeutic effects of dexamethasone in multiple myeloma. Conclusions: Selinexor increased the nuclear retention of dexamethasone-activated-GR. The increased GR transcriptional activity induces expression of genes across different pathways leading to inhibition of cell proliferation and increase cancer cell death. We are currently testing the function of several of these genes in the context of Sel-Dex combination. The current finding supports the the previously reported anti cancer activity of Sel-Dex combination in patients with heavily pretreated relapsed/refractory multiple myeloma. Disclosures Kashyap: Pharma: Employment. Klebanov:Karyopharm Therapeutics Inc: Employment. Lee:Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm Therapeutics: Employment.

[Background] Survivin is a member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. However, the effect of this agent on multiple myeloma (MM) cells remains unclear. [Materials & Methods] Five human MM cell lines, RPMI8226, U266, KMS20, KMS28PE, and KMS34 were used in this study. Cell proliferation and cell death were evaluated by MTT assay and by flow cytometric analysis with annexin V/PI staining. Gene and protein expressions were analyzed with quantitative PCR (qPCR) and immunoblot, respectively. For proteasome inhibitory assay, cells were treated with YM155 and/or proteasome inhibitor MG132 for 6 hours. [Results] YM155 inhibited cell proliferation of these cells in a dose- and time-dependent manner. Annexin V assay showed that YM155 induced apoptosis in these cells. To better understand these effects of YM155 on MM cells, we evaluated the intracellular signaling and apoptosis-associated protein status. Immunoblot analyses showed that YM155 reduced not only survivin but also Mcl-1 and XIAP expressions. We also observed the activation of caspase-3 and PARP in YM155-treated cells, indicating that YM155 induces caspase-dependent apoptosis. In contrast, YM155 did not affect phosphorylation status of Erk1/2 and STAT3. Interestingly, YM155 suppressed c-Myc and IRF4 protein expressions, both of which are recognized as an important transcription factor in the pathogenesis of MM. In addition, qPCR assay showed that YM155 treatment did not reduce c-Myc mRNA level. On the other hand, proteasome inhibitor prevented the suppression of c-Myc expression by YM155 treatment, indicating a proteasomal degradation of c-Myc by YM155. [Conclusion] YM155 suppresses cell proliferation and survival in MM cells in part via not only inhibiting anti-apoptotic proteins such as survivin, Mcl-1 and XIAP but also suppressing c-Myc oncoprotein expression. Further study is needed to clarify the molecular mechanism of c-Myc degradation induced by YM155. Our results may provide a new concept in c-Myc-targeting therapy for MM because c-Myc oncoprotein have been considered undruggable. Disclosures Ishida: Bristol-Myers Squibb: Honoraria.

In a phase 1/2 two-arm trial, 54 patients with myeloma received autografts followed by ex vivo anti-CD3/anti-CD28 costimulated autologous T cells at day 2 after transplantation. Study patients positive for human leukocyte antigen A2 (arm A, n = 28) also received pneumococcal conjugate vaccine immunizations before and after transplantation and a multipeptide tumor antigen vaccine derived from the human telomerase reverse transcriptase and the antiapoptotic protein survivin. Patients negative for human leukocyte antigen A2 (arm B, n = 26) received the pneumococcal conjugate vaccine only. Patients exhibited robust T-cell recoveries by day 14 with supraphysiologic T-cell counts accompanied by a sustained reduction in regulatory T cells. The median event-free survival (EFS) for all patients is 20 months (95% confidence interval, 14.6-24.7 months); the projected 3-year overall survival is 83%. A subset of patients in arm A (36%) developed immune responses to the tumor antigen vaccine by tetramer assays, but this cohort did not exhibit better EFS. Higher posttransplantation CD4+ T-cell counts and a lower percentage of FOXP3+ T cells were associated with improved EFS. Patients exhibited accelerated polyclonal immunoglobulin recovery compared with patients without T-cell transfers. Adoptive transfer of tumor antigen vaccine-primed and costimulated T cells leads to augmented and accelerated cellular and humoral immune reconstitution, including antitumor immunity, after autologous stem cell transplantation for myeloma. This study was registered at www.clinicaltrials.gov as NCT00499577.

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

Polyploidization of megakaryocytes (MKs), the platelet precursors, occurs by endomitosis, a mitotic process that fails at late stages of cytokinesis. Expression and function of Aurora B kinase during endomitosis remain controversial. Here, we report that Aurora B is normally expressed during the human MK endomitotic process. Aurora B localized normally in the midzone or midbody during anaphase and telophase in low ploidy megakaryocytes and in up to 16N rare endomitotic MKs was observed. Aurora B was also functional during cytokinesis as attested by phosphorylation of both its activation site and MgcRacGAP, its main substrate. However, despite its activation, Aurora B did not prevent furrow regression. Inhibition of Aurora B by AZD1152-HQPA decreased cell cycle entry both in 2N to 4N and polyploid MKs and induced apoptosis mainly in 2N to 4N cells. In both MK classes, AZD1152-HQPA induced p53 activation and retinoblastoma hypophosphorylation. Resistance of polyploid MKs to apoptosis correlated to a high BclxL level. Aurora B inhibition did not impair MK polyploidization but profoundly modified the endomitotic process by inducing a mis-segregation of chromosomes and a mitotic failure in anaphase. This indicates that Aurora B is dispensable for MK polyploidization but is necessary to achieve a normal endomitotic process.

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds, we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia, myeloma, and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore, oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition, oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models, thereby establishing its ability to target leukemia stem cells. Mechanistically, CPX bound intracellular iron, and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance, CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus, in summary, CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore, CPX could be rapidly repurposed for the treatment of malignancies, including leukemia and myeloma.