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Two classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3-internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor-κB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencing-defined poor-prognostic cancer hotspot mutations, and CD34(+)/CD38(-)/CD123(+) populations, but not normal CD34(+) progenitors. Finally, combined treatment markedly reduced tumor burden and significantly prolonged animal survival (P < .0001) in AML xenograft models with negligible toxicity, accompanied by pharmacodynamic effects observed in vitro. Collectively, these findings argue that MLN4924 and belinostat interact synergistically by reciprocally disabling the DDR in AML/MDS cells. This strategy warrants further consideration in AML/MDS, particularly in disease with unfavorable genetic aberrations.

Bim contributes to resistance to various standard and novel agents. Here we demonstrate that Bim plays a functional role in bortezomib resistance in multiple myeloma (MM) cells and that targeting Bim by combining histone deacetylase inhibitors (HDACIs) with BH3 mimetics (eg, ABT-737) overcomes bortezomib resistance. BH3-only protein profiling revealed high Bim levels (Bim(hi)) in most MM cell lines and primary CD138(+) MM samples. Whereas short hairpin RNA Bim knockdown conferred bortezomib resistance in Bim(hi) cells, adaptive bortezomib-resistant cells displayed marked Bim downregulation. HDACI upregulated Bim and, when combined with ABT-737, which released Bim from Bcl-2/Bcl-xL, potently killed bortezomib-resistant cells. These events were correlated with Bim-associated autophagy attenuation, whereas Bim knockdown sharply increased autophagy in Bim(hi) cells. In Bim(low) cells, autophagy disruption by chloroquine (CQ) was required for HDACI/ABT-737 to induce Bim expression and lethality. CQ also further enhanced HDACI/ABT-737 lethality in bortezomib-resistant cells. Finally, HDACI failed to diminish autophagy or potentiate ABT-737-induced apoptosis in bim(-/-) mouse embryonic fibroblasts. Thus, Bim deficiency represents a novel mechanism of adaptive bortezomib resistance in MM cells, and Bim-targeting strategies combining HDACIs (which upregulate Bim) and BH3 mimetics (which unleash Bim from antiapoptotic proteins) overcomes such resistance, in part by disabling cytoprotective autophagy.

Introduction: Emerging evidence supports that cytogenetic abnormalities (CAs) drive myelomagenesis and heterogeneity (e.g., clinical presentation, response to therapy, and prognosis) of multiple myeloma (MM). Diversity of CAs, including primary (IgH translocations and trisomies) and secondary CAs (copy number abnormalities) argues that MM is not a "single disease". Thus, identification of CAs at diagnosis is essential for risk stratification, guiding treatment, and prognostic estimation in daily practice. However, although frequency, configuration, and significance of CAs have been well documented in the Western countries, this information is lacking in the Asian population. To this end, a multi-center retrospective analysis was carried out to examine epidemiology and prognostic significance of CAs alone or in combination in a cohort of Chinese patients with newly-diagnosed MM (NDMM). Materials and Methods: A total of 1015 NDMM patients who had the baseline information of CAs detected by FISH at four institutes nationwide were included. According to the IMWG consensus updated in 2016, 1q gain, del(17p), t[4;14], and t[14;16] were defined as high-risk CAs (HRCAs), while another HRCA t[14;20] was not tested routinely in a majority of these patients. In addition, del(1p) and del(13q14) were considered as an adverse CA. According to the mSMART3.0 proposed by Mayo Clinic in 2018, double-hit (DHMM) and triple-hit MM (THMM) were defined as co-occurrence of 2 or >= 3 HRCAs, respectively. The Kaplan-Meier approach was used to estimate progression-free survival (PFS) and overall survival (OS). Results: In this cohort, the median age of 1015 patients was 61 years; 63.5% were male. The type of IgG, light chain, IgA, IgD, non/oligosecretory, IgM, or IgE accounted for 42.9%, 26.2%, 24.5%, 3.4%, 2.5%, 0.4%, and 0.1%, respectively. Del(13q) (46.4%) and 1q gain (46.1%; 3 copies = 73.8%, >= 4 copies = 26.2%) represented the most common CAs, followed by t(4;14) (14.0%), t(11;14) (11.8%), del(1p) (11.5%), del(17p) (9.9%; 20-50% cells = 35.6%, > 50% cells = 64.4%), and t(14;16) (5.1%). While none of these CAs was detected in 23.8% of cases, the frequency of patients who carried 1 - 5 CAs was 31.9%, 28.0%, 13.4%, 2.0%, and 0.9%, respectively. In the 1q+ cases, 36.4% patients carried second CA(s), including del(13q) (61.1%), t(4;14) (20.3%), del(1p) (14.8%), del(17p) (10.7%), t(11;14) (10.4%), and t(14;16) (8.1%). In the del(17p) cases, 57.5% patients had additional CA(s), including del(13q) (75.2%), 1q gain (49.5%), del(1p) (21.6%), t(4;14) (19.6%), t(14;16) (8.7%), and t(11;14) (2.2%). In the cases bearing IgH translocations, 26.0% patients also carried other CA(s), including del(13q) and 1q gain (61.9% for each), and del(17p) (9.3%). In the cases harboring HRCAs, the percentage of patients who carried 1 - 4 HRCAs was 70.2%, 24.8%, 3.7%, and 1.1%, respectively. Overall, 14.3% and 2.9% patients had DHMM or THMM, of whom 65.0%, 18.0%, 12.0%, and 5.0% had 2 - 5 HRCAs, respectively. While there was no significant difference in PFS between the cases carrying 1 and 2 CAs (P = 0.209), the patients who had 3 or more CAs displayed a sharp reduction in median PFS (P = 0.022 and P = 0.003 for 3 vs 1 or 2 CAs). Although multiple CAs was associated with shorter median OS, no statistical significance was observed for each comparison (P > 0.05). However, patients who carried >= 2 HRCAs had significantly shorter median PFS (12.1 months; P = 0.0004) and OS (29.3 months; P = 0.027) than those who had one single HRCA (32.2 and 65.6 months for median PFS and OS). Conclusion: In comparison with the Western countries, the incidence of secondary HRCAs (e.g., 1q gain and del(17p)) is relatively higher in Chinese patients at diagnosis, while the standard-risk CA such as t(11;14) is clearly less frequent. The proportion of Chinese patients who carry multiple CAs (up to 5) or HRCAs appear to be greater as well. In this context, patients carrying two or more HRCAs, so called DHMM or THMM, exhibit significantly worse outcome than those carrying only one HRCA. Together, this study builds up an up-to-date profile of CAs for Chinese patients, which might lay a foundation for revising the criteria for risk stratification and the guideline for treatment that is more feasible and practicable in China. It also provides the information about frequency and configuration of MM-driven CAs, which might be more relevant to the Asian population. Disclosures Kumar: Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Introduction: Interferon regulatory factor 4 (IRF4) is a transcription factor involved in B cell activation and differentiation into plasma cells, as well as B-cell lineage neoplasia. Diverse genetic abnormalities and dysregulated expression of IRF4 have been documented in various hematologic malignancies, including multiple myeloma (MM). IRF4 is also identified as a major downstream target of CRBN/IKZF to regulate MYC expression, representing a primary mechanism underlying anti-MM action of IMiDs. However, it remains unclear how the expression and function of IRF4 are regulated in MM cells. Materials and Methods: DNAseq, whole exome RNAseq (WES), and label-free analyses of quantitative proteomics and post-translational modifications (PTMs) mapping were conducted to examine genetic aberrations, transcriptional dysregulation, or protein and PTM alterations in primary patient samples and drug-naive vs -resistant cell lines. Flow cytometry, qPCR, and Western blot analysis were utilized to monitor apoptosis or mRNA and protein levels of target genes. Stable transfection with wild-type or mutated IRF4 was carried out to evaluate its function. Analysis of the MM genome-wide GEP databases (R2: Genomics Analysis and Visualization Platform) was performed to validate the clinical significance of IRF4 and its target genes in MM patients. Results: MM cells that acquired bortezomib (btz) resistance (PS-R) exhibited a marked increase in protein levels of multiple key components of the canonical and non-canonical NF-κB signaling cascades, in association with increased NF-κB transcriptional activity. Pharmacological inhibition of NF-κB using several compounds (e.g., Bay 11-7082, parthenolide, triptolide) significantly increased btz sensitivity in PS-R cells and their parental U266 cells, as well as other MM cell lines. These events were accompanied by down-regulation of various genes, including NF-κB-dependent genes and those whose relationship with NF-κB remains uncertain. The latter included IRF4, MYC, MCL1, BIRC2/cIAP1, SQSTM1/p62, among others. Of note, overexpression of wild-type IRF4 sharply reduced sensitivity of drug-naive cells to btz alone or in combination with NF-κB inhibitors. However, IRF4 overexpression did not prevent inactivation of NF-κB and down-regulation of the NF-κB-dependent genes (e.g., Bcl-xL, TNFAIP3/A20), suggesting that IRF4 acts downstream of NF-κB. Surprisingly, IRF4 overexpression failed to restore expression of MYC, a well-established target of IRF4, while largely reversed expression of cIAP1 and Mcl-1 at both mRNA and protein levels. Moreover, inhibitors of either IAP (birinapant/TL32711) or Mcl-1 (S63845) overcame btz-resistance conferred by IRF4 overexpression. DNAseq unveiled multiple IRF4 SNPs located in introns and exons (both 3'-UTR and coding region) of chromosome 6. WES revealed that IRF4 was highly expressed in primary bone marrow and extramedullary samples of MM patients (2~10 folds over normal donors). But no difference at mRNA and protein levels of IRF4 was observed between drug-naive and -resistant cell lines. The global PTM mapping identified two lysine residues (K59 and K399) that were highly ubiquitinated in PS-R cells (p = 0.0447 and p = 0.0004, vs U266 cells). Interestingly, ectopic expression of IRF4 mutants (e.g., K59N, K123R) lost the cytoprotective capability of their wild-type counterpart, in association with failure to reverse down-regulation of cIAP1 and Mcl-1. This observation argues that these point mutations are loss-of-function, which might explain better prognosis of patients carrying IRF4 mutations (including K59N and K123R) than wild-type IRF4 as reported previously. Last, the MM GEP database analysis validated the correlation between IRF4 and Mcl-1 or cIAP expression, as well as the adverse effect of their dysregulation on overall survival of MM patients. Conclusion: IRF4 is highly expressed in MM cells, likely via an NF-κB-dependent mechanism. IRF4 acts downstream of the NF-κB signaling pathways, which in turn directly targets cIAP1 and Mcl-1, rather than MYC, to confer drug resistance. IRF4 could be mutated in MM patients, while the point mutations, at least at certain lysine sites, seem to be loss-of-function and thus predict better outcome. Together, these findings suggest that the oncogenic function of IRF4 might be governed at genetic, transcriptional, and PTM levels in MM cells. Disclosures Kumar: Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

Introduction: Histone lysine methylation, a reversible event dynamically and reciprocally regulated by lysine methyltransferases (KMTs) and demethylases (KDMs), represents one of the major epigenetic mechanisms for regulation of chromatin remodeling and gene expression re-programming. The KDM4 family, which belongs to the Jumonji C (JmjC)-domain-containing proteins (JMJDs), consists of five members, including KDM4A-E that demethylate H3K9me2/3 and/or H3K36me2/3 in a Fe2+- and α-ketoglutarate-dependent manner. KDM4 proteins are involved in various cellular processes such as gene transcription and translation, DNA replication, DNA repair, apoptosis, and stem cell renewal. Notably, increasing evidence implicates KDM4 dysregulation in promoting genomic instabilities and oncogenesis, thereby which is considered as a potential target for emerging cancer epigenetic therapy. Although KDM4A, a member of the KDM4 family, has been widely studied in many solid tumors including breast, prostate, bladder cancer, its role in hematopoietic malignancies, including multiple myeloma (MM), remains unknown. Materials and Methods: Human MM cell lines (U266, RPMI8226, H929, OPM-2) were employed. After exposed to hypoxia (or the chemical hypoxia mimetic lactic acid) and anti-MM agents (e.g., bortezomib/Btz), cells were analyzed by flow cytometry, qPCR, Western blot to monitor apoptosis, cell cycle, proliferation (Ki67), DNA double-strand break/DSB (γH2A.X), expression of 1q21 and anti-apoptotic genes, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down KDM4A for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary MM samples to link KDM4A expression to disease progression and therapeutic response. Results: Analysis of the MM genome-wide GEP databases revealed that KDM4A mRNA was significantly up-regulated in MGUS and MM, but not SMM, compared to normal control, as well as in relapsed MM, compared to newly-diagnosed MM. To our surprise, KDM4A expression rather favored overall survival of MM patients, including those carrying 1q21 gain in whom KDM4A expression was indeed lower than those who did not have this high risk cytogenetic abnormality. Moreover, KDM4A expression correlated adversely with expression of 1q21 genes (e.g., CKS1B, MCL1, PSMD4, ARNT). Whereas basal KDM4A protein level was moderately but clearly higher in MM cell lines carrying 1q21 gain or acquired drug resistance than their counterparts, exposure to hypoxia or lactic acid (but not cobalt chloride) resulted in marked KDM4A up-regulation, accompanied by NF-κB and HIF pathway activation. However, while NF-κB inhibition and to a lesser extent ARNT/HIF-1β knockdown led to a robust increase in hypoxia-induced KDM4A expression, shRNA knockdown or pharmacological inhibition of KDM4A triggered NF-κB activation and HIF expression, as well as up-regulated anti-apoptotic proteins (e.g., Mcl-1, TNFAIP3/A20, CKS1B), in association with increased H3K36me3 rather than H3K9me3. Furthermore, KDM4A knockdown or inhibition sharply diminished Btz lethality and overrode hypoxia-mediated cytoprotection. Interestingly, KDM4A knockdown also increased MM cell proliferation, promoted S phase entry, and attenuated Btz-induced DSB. Last, IHC of sequential bone marrow biopsies revealed that while KDM4A protein was relatively low at diagnosis, its level was markedly increased when patients achieved CR and then fell to the baseline low level at relapse. Conclusion: KDM4A/JMJD2A, a lysine demethylase that has been recognized as an pro-oncogenic protein via its epigenetic and/or non-epigenetic properties, is identified for the first time as a potential tumor suppressor in MM, particularly in a high risk subtype carrying 1q21 gain. Whereas KDM4A is expressed in MM and can be further induced by hypoxia that naturally exists in bone marrow niche, it seems to play multiple inhibitory roles in cell growth, cell cycle, DNA repair, and drug resistance by suppressing expression of oncogenic and anti-apoptotic genes (especially 1q21 genes), likely via H3K36me3 demethylation, and antagonizing NF-κB and HIF activation. These findings suggest that in contrast to its pro-oncogenic role in certain solid tumors, KDM4A might instead act as a tumor suppressor in MM. This work was supported by NNSFC (81471165, 81670189, and 81670190). Disclosures Kumar: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Plasticity is one of the hallmarks of macrophages, an essential component of innate and adaptive immunity. In response to various stimuli, macrophages can differentiate or polarize to either pro-inflammatory M1 or anti-inflammatory M2 phenotype, which determines whether inflammation is initiated and promoted or terminated and resolved. Of note, the phenotype of polarized M1-M2 macrophages may be reversed in certain circumstances, providing an opportunity to treat inflammatory disorders (e.g., atherosclerosis) and inflammation-related diseases (e.g., cancer) by targeting macrophage polarization. Emerging evidence supports that transcriptional regulation play an important role in polarization and function of macrophages via reprogramming expression of pro- versus anti-inflammatory genes. However, despite well-established cross-talk between two major transcriptional factors, NF-κB and hypoxia-inducible factor (HIF), it remains unclear whether NF-κB interacts with HIFs (particularly HIF-1β, a regulatory subunit of the active HIF complex) in reprogramming macrophages. Here, we investigated the mechanism underlying macrophage activation induced by oxidized low density lipoprotein (oxLDL), a central event of uncontrolled inflammation in atherosclerosis. Materials and Methods: The murine macrophage cell line RAW264.7 and human THP-1 cell line-derived macrophages were employed. After exposed to oxLDL, cells were analyzed by qPCR, Western blot, flow cytometry (Cytometric Bead Array, CBA), and ELISA analyses to monitor expression of M1 and M2 markers and related cytokines, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down expression of target genes for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary samples obtained from healthy donors (n = 10) and patients with coronary heart disease (n = 22) and stroke (n = 11). Results: Exposure to oxLDL triggered M1 polarization of murine and human macrophages, characterized by expression of iNOS and robust production of M1 pro-inflammatory cytokines (e.g., TNF-α, MCP-1, IL-1β, IL-6) but not M2 anti-inflammatory cytokines (e.g., IL-10, TGF-β). In contrast, protein level of the M2 marker Arg1 was clearly decreased after treated with oxLDL. Notably, exposure of macrophages to oxLDL resulted in markedly increased expression of HIF-1α and -1β, in association with activation of both canonical and non-canonical NF-κB pathways. Functionally, whereas inhibition of NF-κB activation by the IKK inhibitor parthenolide almost completely prevented M1 polarization and promoted M2 polarization, knockdown of HIF-1β by shRNA also largely reversed macrophage polarization from M1 to M2 after exposed to oxLDL. These results were confirmed in human macrophages differentiated by PMA from primary peripheral blood monocytes obtained from patients with coronary heart disease or ischemic stroke, and normal donors. These events were accompanied by a clear reversal of oxLDL-induced morphological changes of macrophages. Mechanistically, inhibition of NF-κB activation dramatically diminished expression of HIF-1α and -1β induced by oxLDL. However, while shRNA knockdown of HIF-1β sharply blocked HIF-1α expression in macrophages exposed to oxLDL, it failed to impair activation of NF-κB. These findings indicate that oxLDL-induced HIF-1β expression is dependent on NF-κB activation, which in turn activates the HIF pathway via HIF-1α up-regulation probably by stabilizing HIF-1α protein. Conclusion: HIF-1β (encoded by ARNT) is identified for the first time as a novel target that reprograms M1-M2 polarization of macrophages, at least after exposure to oxLDL, a risk factor of atherosclerosis. HIF-1β is further demonstrated to act as downstream of NF-κB to induce activation of the HIF pathway. Importantly, these findings suggest that HIF-1β might serve as a therapeutic target for the treatment of inflammatory disorders such as atherosclerosis, and probably immune diseases and cancer as well. Disclosures No relevant conflicts of interest to declare.

Interactions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl+ leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34+ chronic myelogenous leukemia (CML) cells and Ba/F3 cells bearing various Bcr/Abl mutations (ie, T315I, E255K, and M351T), as well as IM-resistant K562 cells exhibiting Bcr/Abl-independent, Lyn-dependent resistance. These events were associated with inactivation and down-regulation of wild-type (wt) and mutated Bcr/Abl (particularly T315I). Moreover, treatment with MK-0457 resulted in accumulation of cells with 4N or more DNA content, whereas coadministration of vorinostat markedly enhanced aurora kinase inhibition by MK-0457, and preferentially killed polyploid cells. Furthermore, vorinostat also interacted with a selective inhibitor of aurora kinase A and B to potentiate apoptosis without modifying Bcr/Abl activity. Finally, vorinostat markedly induced Bim expression, while blockade of Bim induction by siRNA dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. Together, these findings indicate that vorinostat strikingly increases MK-0457 activity against IM-sensitive and -resistant CML cells through inactivation of Bcr/Abl and aurora kinases, as well as by induction of Bim.

The role of Bim in synergistic interactions between UCN-01 and MEK1/2 inhibitors in human multiple myeloma cells was investigated. Exposure of U266 or RPMI8226 cells to UCN-01 resulted in ERK1/2 activation-associated BimEL phosphorylation/down-regulation, events abrogated by MEK1/2 inhibitors. Enforced activation of ERK1/2 by transfection with constitutively active MEK1 diminished the capacity of PD98059 but not PD184352 to block UCN-01–mediated BimEL phosphorylation and to potentiate apoptosis. Cotreatment with MEK1/2 inhibitors increased the association of BimEL with both Bcl-2 and Bcl-xL in UCN-01–treated cells, leading to Bax/Bak conformational change and Bax mitochondrial translocation. Down-regulation of BimEL by shRNA substantially diminished UCN-01/MEK inhibitor-mediated Bax/Bak activation and apoptosis. Furthermore, transfection of cells with S65A Bim, a mutant resistant to UCN-01–mediated phosphorylation, significantly sensitized cells to UCN-01 lethality. Conversely, ectopic expression of either Bcl-2 or Bcl-xL did not alter UCN-01/MEK1/2 inhibitor-mediated modifications in BimEL phosphorylation but largely prevented cell death. Finally, IL-6 or IGF-1 failed to prevent MEK1/2 inhibitors from blocking UCN-01–induced BimEL phosphorylation/degradation or cell death. Collectively, these findings argue that UCN-01–mediated ERK1/2 activation leads to BimEL phosphorylation/inactivation, resulting in cytoprotection, and that interference with these events by MEK1/2 inhibitors plays a critical role in synergistic induction of apoptosis by these agents.

Interactions between UCN-01 and HMG-CoA reductase inhibitors (ie, statins) have been examined in human leukemia and myeloma cells. Exposure of U937 and U266 cells to minimally toxic concentrations of UCN-01 and various statins (eg, lovastatin, simvastatin, or fluvastatin) dramatically increased mitochondrial dysfunction, caspase activation, and apoptosis. Comparable effects were observed in other leukemia and myeloma cell lines as well as in primary acute myeloid leukemia (AML) blasts but not in normal hematopoietic cells. Potentiation of UCN-01 lethality by lovastatin was associated with disruption of Ras prenylation and activation. These events were significantly attenuated by farnesyl pyrophosphate (FPP) but not by geranylgeranyl pyrophosphate (GGPP), implicating perturbations in farnesylation rather than geranylgeranylation in synergistic interactions. Coexposure to statins and UCN-01 resulted in inactivation of ERK1/2 and Akt, accompanied by JNK activation. U266 cells ectopically expressing JNK1-APF, a dominant negative JNK1 mutant, displayed significantly reduced susceptibility to lovastatin/UCN-01–mediated lethality. Moreover, transfection of U266 cells with constitutively activated H-Ras (Q61L) attenuated ERK1/2 inactivation and dramatically diminished the lethality of this regimen. Collectively, these findings indicate that HMG-CoA reductase inhibitors act through a Ras farnesylation-associated mechanism to induce signaling perturbations, particularly prevention of Ras and ERK1/2 activation, in UCN-01–treated cells, resulting in the synergistic induction of cell death.

Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and the proteasome inhibitor bortezomib were examined in Bcr/Abl+ human leukemia cells. Coexposure of K562 or LAMA84 cells to subtoxic concentration of flavopiridol (150-200 nM) and bortezomib (5-8 nM) resulted in a synergistic increase in mitochondrial dysfunction and apoptosis. These events were associated with a marked diminution in nuclear factor κB (NF-κB)/DNA binding activity; enhanced phosphorylation of SEK1/MKK4 (stress-activated protein kinase/extracellular signal-related kinase 1/mitogen-activated protein kinase kinase 4), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK); down-regulation of Bcr/Abl; and a marked reduction in signal transducer and activator of transcription 3 (STAT3) and STAT5 activity. In imatinib mesylate-resistant K562 cells displaying increased Bcr/Abl expression, bortezomib/flavopiridol treatment markedly increased apoptosis in association with down-regulation of Bcr/Abl and BclxL, and diminished phosphorylation of Lyn, Hck, CrkL, and Akt. Parallel studies were performed in imatinib mesylate-resistant LAMA84 cells exhibiting reduced expression of Bcr/Abl but a marked increase in expression/activation of Lyn and Hck. Flavopiridol/bortezomib effectively induced apoptosis in these cells in association with Lyn and Hck inactivation. The capacity of flavopiridol to promote bortezomib-mediated Bcr/Abl down-regulation and apoptosis was mimicked by the positive transcription elongation factor-b (P-TEFb) inhibitor DRB (5,6-dichloro 1-β-d-ribofuranosylbenzinida-sole). Finally, the bortezomib/flavopiridol regimen also potently induced apoptosis in Bcr/Abl- human leukemia cells. Collectively, these findings suggest that a strategy combining flavopiridol and bortezomib warrants further examination in chronic myelogenous leukemia and related hematologic malignancies. (Blood. 2004;104:509-518)

Interactions between pharmacologic NF-κB inhibitors (eg, Bay 11-7082, SN-50) and the checkpoint abrogator UCN-01 have been examined in human multiple myeloma (MM) cells. Exposure of U266 cells to Bay 11-7082 (Bay) in combination with UCN-01 resulted in the abrogation of NF-κB/DNA binding activity and the synergistic induction of apoptosis. Comparable synergism was observed in other MM cell lines and patient-derived CD138+ cells and between an inhibitory peptide of NF-κB (SN50) and UCN-01. Bay/UCN-01-mediated lethality involved mitochondrial dysfunction, caspase cleavage, and poly adenosine diphosphate-ribose polymerase (PARP) degradation. Although Bay modestly blocked UCN-01-induced extracellular signal-regulated kinase (ERK) phosphorylation, coadministration activated c-Jun N-terminal kinase (JNK) and cdc2/cdk1 and down-regulated Mcl-1, XIAP, and Bcl-xL. Transfection with a constitutively activated mitogen-activated protein kinase kinase (MEK1)/green fluorescent protein (GFP) construct failed to block apoptosis induced by Bay/UCN-01 but significantly attenuated MEK inhibitor (U0126)/UCN-01-induced lethality. Inhibiting JNK activation with SP600125 or D-JNKI1 peptide markedly reduced Bay/UCN-01-mediated mitochondrial dysfunction and apoptosis and the down-regulation of Mcl-1, XIAP, and Bcl-xL but not of cdc2/cdk1 activation. Stable transfection of cells with dominant-negative caspase-9 dramatically diminished Bay/UCN-01 lethality without altering JNK or cdc2/cdk1 activation. Neither interleukin-6 (IL-6)- nor fibronectin-mediated adherence conferred resistance to Bay/UCN-01-induced apoptosis. Together, these findings suggest that a strategy combining UCN-01 with disruption of the IκB kinase (IKK)/IκB/NF-κB pathway warrants attention in MM. (Blood. 2004;103:2761-2770)

Introduction: Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired-resistance in MM. Materials and Methods: PI-resistant myeloma cell lines, RPMI8226-B25 and U226 PSR, and their respective parental cell lines RPMI8226 and U266, were treated both in vitro with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. In vivo studies using U266 and U266PSR tumors were performed in NOD/SCID-gamma (NSG) mice. Mice were treated with selinexor/bortezomib and single agents. Bone marrow biopsies from refractory myeloma patients were treated ex vivo with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. Mechanistic studies included NFkB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in selinexor/bortezomib treated MM cells. Results: We found that selinexor restored sensitivity of PI-resistant RPMI8226-B25 and U266PSR MM cells to bortezomib (P = 0.00055) and carfilzomib (P = 0.0017). Bortezomib, when combined with selinexor reduced U266 MM tumor growth versus single-agent bortezomib (P = 0.022) in NSG mice. NSG mice challenged with PI-resistant U266PSR MM tumors also had reduced tumor growth with selinexor/bortezomib as compared to single agent bortezomib (P = 0.0006). Combining bortezomib and selinexor improved survival in mice with U266 MM tumors (P = 0.0072) and PI-resistant U266PSR when compared to single-agent bortezomib (P = 0.0072). Myeloma cells from PI-refractory MM patients (n=14) were sensitized by selinexor to bortezomib (P = 0.002) and carfilzomib (P = 0.001) without affecting non-myeloma cells. Immunofluorescence microscopy of PI-resistant human MM cell lines found a greater than 212% increase in IkBα when compared to untreated cells (confirmed by Western blot). A similar increase in IkBα immunofluorescence was found in newly diagnosed, relapsed and refractory patient MM cells. ImageStream analyses of MM cells showed an increase in total and nuclear IkBα from selinexor/bortezomib exposure. Proximity-ligation assays showed that IkBα-NFkB-complexes were increased 12-fold in bortezomib/selinexor treated MM cells. IkBα knockdown abrogated selinexor/bortezomib induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFkB transcriptional activity in addition to a reduction of NFkB induced IAP-1, IAP-2, BCL-2, cyclin D2 and c-myc protein expression.. Conclusions: Selinexor, when used with bortezomib or carfilzomib has the potential to overcome proteasome-inhibitor drug-resistance in MM. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Selinexor, an orally active selective inhibitor of XPO1-mediated nuclear export (SINE), is currently undergoing phase I/II studies in a variety of indications, including a combination with carfilzomib, in both relapsed and refractory MM patients (NCT02199665). The results presented in this study support combinatorial clinical trials in relapsed and refractory MM that utilize PI therapies. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Shain:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Signal Genetics: Research Funding; Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder.

Introduction:Although the macrolide antibiotic clarithromycin (CAM, or Biaxin) has only minimal single agent activity in MM, the regimens with addition of CAM to IMiDs and steroids, including BLT-D (Biaxin/low-dose thalidomide/dexamethasone [Dex]), BiRD (Biaxin/Revlimid [lenalidomide, Lena]/Dex), t-BiRD (thalidomide/BiRD), Car-BiRD (carfilzomib/BiRD), and ClaPD (CAM/pomalidomide/Dex), appear highly effective in treatment of newly-diagnosed and probably relapsed/refractory multiple myeloma (MM). In this context, two phase 3 trials are currently ongoing to evaluate the efficacy of BiRD vs. RD in newly-diagnosed MM in the United States and Europe, respectively. Of note, recent findings also suggests that addition of CAM to RD might overcome resistance to RD. However, despite increasing clinical evidence for its promising activity, the exact mechanism for such a combination strategy still remain largely unclear. Here, we investigated the mechanisms of action underlying the interaction between CAM and Lena and their capability to overcome drug resistance in MM cells, focusing on the cereblon (CRBN)/IKZF1,3/IRF4/Myc signaling cascade, recently identified as the novel target of IMiDs, Materials and Methods: To test our hypothesis whether and how BiRD overcomes resistance to RD (Lena/Dex), human MM cells lines employed in this study included Dex-sensitive (MM.1S) vs -resistant (MM.1R) cells, drug-naïve RPMI8226 cells vs their Lena-resistant (R10R) or bortezomib (Btz)-resistant counterparts (DR), as well as primary CD138+MM cells isolated from bone marrow samples of newly-diagnosed and relapsed/refractory patients who had received prior IMiDs (including Lena) or Btz. Cells were exposed (72 hr) to CAM (50-100 mg/ml) ± Lena (1-10 mM), after which the CCK-8 assay and flow cytometry with annexin V/7AAD staining were performed to monitor cell viability and apoptosis, respectively. Mechanistic studies included Western blot analyses of the CRBN/IKZF1,3/IRF4/Myc signaling pathway, as well as the apoptotic caspase cascade. Cell cycle was also assessed by flow cytometry. Results: Whereas Lena (1-10 mM) had almost no direct effects on cell viability, CAM (≥ 100 mg/ml) displayed a dose-dependent toxicity in various MM cell lines. Notably, subtoxic concentrations of CAM (e.g., 50 mg/ml) significantly potentiated lethality of Lena in MM.1S (CI value = 0.40-0.86, indicating synergism). Significantly, this effect was even more robust in Dex-resistant MM.1R cells. These events were associated with marked activation of caspase 3, 8, and 9 and PARP cleavage, accompanied by down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL. While add-on of CAM significantly increased lethality of Lena in RPMI8226 cells, combined treatment was strikingly more effective against Lena-resistant R10R cells. In addition, Btz-resistant RPMI8226 cells were also more sensitive to both CAM alone and in combination with Lena, compared to parental RPMI8226 cells. Consistently, markedly enhanced cell killing by the combination was also observed in primary CD138+ cells, particularly those obtained from patients relapsed after prior IMiDs. Exposure to Lena with or without CAM sharply down-regulated CRBN in MM cells, accompanied by reduced expression of IKZF1, IKZF3, IRF4, and Myc. Interestingly, Lena failed to down-regulate CRBN/IKZF1/IRF4/Myc in Lena-resistant R10R cells, while addition of CAM dramatically resensitized these cells to the action of Lena. Moreover, Lena in the presence or absence of CAM induced cell cycle arrest at G0/G1, in association with marked up-regulation of p21Cip1 and p27Kip1. Last, Lena induced LC3A-II expression (a marker of autophagy), which was clearly increased in the presence of CAM, likely in association with the capability of CAM to impair the late stage process of autophagy e.g., autophagosome clearance by lysosome. Conclusion: Together, these findings indicate that CAM significantly increases the anti-MM activity of Lena in MM cells, especially those resistant to the first-line therapy (e.g., Dex and Btz), and notably overcomes Lena resistance. The mechanisms involves disruption of the CRBN/IKZF1/IRF4/Myc pathway, as well as activation of the apoptotic caspase cascade, induction of cell cycle arrest, and attenuation of autophagy. Collectively, these mechanistic findings support exploring the BiRD regimen in MM, particularly to overcome RD resistance. Disclosures No relevant conflicts of interest to declare.

Purpose: Drug resistance is the greatest obstacle to the successful treatment of multiple myeloma (MM). We investigated whether the clinical XPO1 inhibitor selinexor (KPT-330), when combined with bortezomib or carfilzomib, could overcome proteasome inhibitor (PI) resistance in myeloma. Experimental Design: PI-resistant human MM cell lines 8226-B25 and U266-PSR were treated with the XPO1 inhibitors selinexor or KOS-2464 in combination with bortezomib or carfilzomib and assayed for apoptosis and viability. Mice challenged with PI-resistant human MM cells (U266-PSR) were treated with selinexor +/- bortezomib. CD138+/light-chain+ MM cells from PI-refractory MM patients were treated with selinexor +/- bortezomib or selinexor +/- carfilzomib and assayed for apoptosis. All experiments were compared to the standard of care, bortezomib therapy. IkBα-protein was assayed by Western blot and immunofluorescence microscopy and IkBα-NFkB-complex formation by proximity ligation assay. IkBα protein knockdown in human MM cells by siRNA was performed to determine the mechanism of selinexor inhibitor action. Further analysis of selinexor/bortezomib treatment on intra-cellular protein levels and intra-cellular localization was performed by lysine and N-terminal labeling with six-plex tandem mass tags (heavy isotope) and assayed by LC-MS/MS discovery proteomics. Results: Selinexor in combination with bortezomib or carfilzomib decreased viability and induced apoptosis in PI-resistant MM cells. Resistant MM cell lines were up to 10-fold resistant to single agent bortezomib or carfilzomib when compared to parental cells. The combination of the XPO1 inhibitors selinexor or KOS-2464 sensitized drug resistant cells to bortezomib (P < 0.02) and carfilzomib (P < 0.005) when compared to single agents. Selinexor and bortezomib inhibited PI-resistant MM tumor growth and increased survival with minimal toxicity in NOD/SCID-g mice. Bone marrow mononuclear cells isolated and treated with selinexor or KOS-2464 and bortezomib or carfilzomib from newly diagnosed (n=8), relapsed (n=5), and bortezomib (n=8) and carfilzomib (n=6) refractory MM patient samples were all sensitized by selinexor and KOS-2464 to bortezomib (P < 0.043) and carfilzomib (P < 0.044) as shown by increased apoptosis. Normal, non-myeloma CD138/light-chain double-negative patient cells were not sensitized to apoptosis by XPO1 inhibitors. Immunofluorescence microscopy of IkBα in 8226-B25 PI-resistant cells showed an increase in IkBα after treatment with selinexor/bortezomib as compared with vehicle control or single agent bortezomib or selinexor. Nuclear IκBα was also increased by selinexor treatment. IkBα protein expression was increased by bortezomib (70%) and selinexor (50%) versus control. The selinexor/bortezomib combination increased IkBα protein (212%) as compared to vehicle control or single agent bortezomib or selinexor. Similar results were found in drug-naïve 8226 and U226 cells, as well as PI-resistant 8226-B25 and U225-PSR cells. The increase in nuclear IkBα after selinexor treatment was confirmed by ImageStream flow cytometry. Selinexor/bortezomib therapy significantly increased IkBα-NFkB-complexes in PI-resistant MM cells. Selinexor in combination with bortezomib increased proximity co-localization of NFkB and IkBα without affecting XPO1 protein expression after 4 hours of drug treatment. Analysis of the number of NFkB-IkBα foci/binding showed that selinexor/bortezomib increased the number of foci in the nucleus versus untreated control or single agent selinexor or bortezomib (P ≤ 0.00077). IkBα knockdown reduced selinexor-induced cytotoxicity in both IM-9 (9.5-fold) and 8226 (12.3 to 25.4-fold) human MM cells. Intracellular protein analysis by heavy isotope labeling and LC-MS/MS showed changes in several signaling pathways including p53, MAPK, VEGF and angiopoietin, IL-1, HMGB1/TLR and APRIL and BAFF as well as those related to NFkB signaling. Conclusion: Selinexor, when used in combination with bortezomib or carfilzomib has the potential to overcome PI drug resistance in MM. Disclosures Kashyap: Pharma: Employment. Landesman:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership.

Purpose Human multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects. Experimental Procedures To test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry. Results Viability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples. Conclusions We found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials. Disclosures Shacham: Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.

Introduction High-dose melphalan chemotherapy with autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma. However, patients eventually develop drug resistance and die from progressive disease despite the introduction of therapies using proteosome inhibitors (PIs) and immunomodulatory drugs (IMIDs). The incurable nature of multiple myeloma clearly demonstrates the need for novel agents and treatments. Here, our aim was to investigate whether the use of XPO1 (exportin 1, CRM1) inhibitors (XPO1i) could sensitize de novo and acquired drug-resistant multiple myeloma cells both in vitro and ex vivo to the alkylating agent melphalan. Materials and Methods Human multiple myeloma cell lines NCI-H929, RPMI-8226, U266 and PBMC controls were treated in vitro with the XPO1i KOS-2464 and the orally available Selective Inhibitor of Nuclear Export (SINE) selinexor (KPT-330) or) +/- melphalan. Multiple myeloma cells were grown at high-density conditions (>3-5x106 cells/mL). High-density multiple myeloma cells have been shown to possess de novo drug resistance. Sensitivity of the XPO1i/melphalan-treated NCI-H929 cells was measured by cell viability assay (CellTiter-Blue). Apoptosis in XPO1i/melphalan-treated NCI-H929, RPMI-8226, and U266 cells was assayed using flow cytometry (activated caspase 3). Proximity ligation assays were performed to assess XPO1-p53 binding in the presence of an XPO1i. Western blots of XPO1i-treated myeloma cells were performed for nuclear and total p53. Drug-resistant U266 (PSR) and 8226 (8226/B25) myeloma cell lines were developed by incremental exposure to bortezomib. PSR cells are able to grow in 15 nM bortezomib and the 8226/B25 in 25 nM. These resistant myeloma cells were treated in vitro with XPO1i +/- melphalan. Sensitivity to therapy was measured by apoptosis and cell viability assay. Multiple myeloma cells isolated from patients with newly diagnosed, relapsed, or refractory disease were treated with XPO1i +/- melphalan and CD138+/light chain+ myeloma cells and assayed for apoptosis. Results Multiple myeloma cell (NCI-H929) viability was decreased synergistically by XPO1i when used in combination with melphalan, as shown by the calculated combinatorial index (CI) values. We examined sequencing of the drugs and found that concurrent treatment with melphalan (10 µM) and selinexor (300 nM) for 48 hours produced the best results (CI value 0.370, n=6). Sequential treatment (selinexor for 24 hours followed by melphalan for an additional 24 hours) or the reverse sequence had slightly less synergy, with CI values of 0.491 (n=9) and 0.565 (n=3), respectively. Normal PBMC control cells were unaffected by XPO1i/melphalan treatment as shown by viability and apoptotic assays. Proximity ligation assay demonstrated that XPO1i blocks XPO1/p53 binding. Western blot showed that the XPO1i treatment of myeloma cells increased nuclear and total p53. Drug-resistant 8226/B25 myeloma cells but not PSR cells were found to be resistant to melphalan when compared to parental cell lines. Both resistant myeloma cell lines were sensitized by XPO1i to melphalan as shown by apoptosis assay (3- to 10-fold). CD138+/light chain+ myeloma cells derived from newly diagnosed, relapsed, and refractory myeloma patients were also sensitized by XPO1 inhibitors to melphalan as demonstrated by apoptotic assays (e.g. activated caspase 3). Conclusions XPO1i synergistically improved the response of de novo and acquired drug-resistant myeloma cells to melphalan in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by XPO1i and the reported activation of p53 by melphalan. Future studies include in vitro experiments using drug-resistant human U266 myeloma cells in NOD-SCID-gamma mice and clinical trials using melphalan in combination with the SINE selinexor. Combination therapies using selinexor and melphalan may significantly improve the treatment of myeloma. Disclosures Kauffman: Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment.

Introduction: Multiple myeloma (MM) is characterized by deregulation of members of the Bcl-2 family of apoptotic regulatory proteins. This has led to the development of BH3-mimetics such as ABT-737 which inhibits Bcl-2/xL but not Mcl-1. Previously, we reported that simultaneous inhibition of Chk1 and MEK1/2 dramatically induced apoptosis in cultured and primary MM cells, including cells resistant to conventional agents, while sparing their normal counterparts (Pei et al., Blood 2007, 2011). Recently, we reported that this strategy circumvented MM cell resistance conferred by overexpression of Mcl-1, an important survival factor in this disease (Pei et al., PLoS One 2014). However, Bcl-2 overexpression confers significant resistance to the Chk1/MEK1/2 inhibition strategy. This raised the possibility that BH3-mimetics targeting Bcl-2 might circumvent this resistance mechanism. The purpose of the present studies was to determine whether BH3-mimetics could overcome such resistance while preserving anti-myeloma selectivity. An additional goal was to employ a new mathematical model to characterize interactions combining three novel agents that coordinately inhibit multiple survival signaling pathways. Methods: Various parental and Bcl-2 or Bcl-xL-over-expressing MM cell lines, as well as primary CD138+ MM cells were employed. ABT-737, the MEK1/2 inhibitor PD184352 (PD), and the Chk1/Wee1 inhibitor (Chk1i) were obtained from Abbott, Millipore and Calbiochem, respectively. Cells were exposed to agents alone or in various combinations for 4 -72 h, after which effects on apoptosis and signaling pathways were determined. Results: Co-administration of ABT-737 potentiated PD/Chk1i-mediated lethality in multiple parental MM cell lines, in association with Mcl-1 down-regulation, Bim up-regulation, and increased DNA damage (ΥH2A.X). Consistent with earlier findings, ectopic expression of Bcl-2 or Bcl-xL protected MM cells from the PD/Chk1i regimen. However, co-administration of ABT-737 significantly restored sensitivity towards PD/Chk1i lethality. Mathematical modeling indicated 3-agent synergistic interactions, particularly in Bcl-2 overexpressing cells. PD/Chk1i exposure inhibited phosphorylation (T705 and S727) of Stat3, another important survival factor for MM cells, while cells expressing constitutively active Stat3 (CA-STAT3) exhibited resistance to this regimen. However, the latter event was reversed by co-exposure to ABT-737. Moreover, combining ABT-737 with PD/Chk1i resulted in release of Bim from anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1, accompanied by Bak and Bax conformational change (activation). Knock-down of Bim by shRNA significantly protected cells from apoptosis induced by the 3-agent combination, indicating a functional role for Bim in anti-MM activity of this regimen. Furthermore, similar interactions, together with down-regulation of pStat3, were also observed in bortezomib-resistant MM cells, as well as in patient-derived CD138+ MM cells. In contrast, the regimen was minimally toxic to normal cord blood CD34+ cells or CD138- bone marrow cells. Finally, co-culture of parental or bortezomib-resistant MM cells with HS-5 stromal cells induced up-regulation of pStat3, while treatment with ABT-737 in combination with PD/Chk1i prevented Stat3 activation and robustly induced apoptosis despite the presence of stromal cells. Conclusion: ABT-737 co-administration synergistically potentiates the lethality of the PD/Chk1i regimen in MM cells, including bortezomib-resistant and primary MM cells, but not in normal hematopoietic progenitors. It also overcomes PD/Chk1i resistance conferred by overexpression of Bcl-2 or Bcl-xL, as well as by microenvironmental factors. Mechanisms responsible for these interactions are likely to be multi-factorial, including inactivation of Stat3, up-regulation of Bim, release of Bim from Bcl-2, Bcl-xL, and Mcl-1, and activation of Bak and Bax. Collectively, these findings demonstrate that combining BH3-mimetics with Chk1/MEK1/2 inhibition circumvents multiple forms of drug resistance in MM cells while exhibiting minimal toxicit toward normal hematopoietic cells. They also argue that a strategy targeting three coordinate survival signaling pathways may be highly effective in killing MM cells, particularly those resistant to current anti-MM therapies. Disclosures No relevant conflicts of interest to declare.

Cell cycle checkpoints play essential roles in the DNA damage response (DDR) which halts cell cycle progression to allow cells to complete DNA replication and repair DNA damage in response to genotoxic insults (e.g., by chemotherapy and ionizing radiation). Wee1 is a dual-specificity protein kinase principally involved in regulation of the G2/M checkpoint that delays mitotic onset by phosphorylating Y15 and deactivating cdc2/Cdk1. AZD1775 is a highly selective small molecule ATP-competitive Wee1 inhibitor currently undergoing clinical evaluation combined with genotoxic agents in solid tumor malignancies (particularly in p53-deficient tumors), based on the concept of mitotic lethality. Recently, Wee1 has been identified as a target in AML, in which AZD1775 potentiates ara-C or mTOR inhibitor lethality. Notably, gene aberrations related to the cell cycle (e.g., 66%) or the DDR (e.g., 41%) are frequent in AML and correlate with poor prognosis (NEJM, 2013). These findings provide a rationale for a novel Wee1-targeting strategy in AML combining AZD1775 with HDAC inhibitors (HDACIs) which also interrupt the DDR and have been granted AML Orphan Drug designation. Here we report that AZD1775 synergistically interacts with HDACIs (e.g., Vorinostat; both Merck via NCI/NIH) in both p53 wild type or mutant AML cell lines, including those carrying poor-prognostic mutations (e.g., FLT3-ITD), NGS-defined patient-derived primary AML specimens, and the CD34+/CD123+/CD38- population enriched for leukemia-initiating progenitors, but spares normal CD34+ hematopoietic cells. Significantly, in marked contrast to Y15 dephosphorylation, AZD1775 triggered pronounced phosphorylation of T14, another critical cdc2/Cdk1 inhibitory site, which might compromise checkpoint-abrogation and thus limit anti-leukemic activity of AZD1775. We determined that T14 phosphorylation most likely stems from compensatory Chk1 activation (e.g., marked phosphorylation of all three critical serine sites, including S296, S317, S345), rather than Myt1 activation, a kinase that phosphorylates T14. Significantly, co-administration of HDACIs substantially diminished Chk1 activation as well as T14 phosphorylation in cells exposed to AZD1775. Consequently, combined AZD1775/HDACI exposure led to dephosphorylation of cdc2/Cdk1 on both Y15 and T14 residues, resulting in full (and “inappropriate”) activation. As a consequence, AZD1775/HDACI co-treatment sharply increased S10 phosphorylation of histone H3 (p-H3), a premature mitotic entry indicator. Flow cytometry analysis revealed that whereas both AZD1775 and HDACIs alone modestly increased the p-H3 mitotic index (MI) at 8 h , co-exposure strikingly increased the p-H3 MI (e.g., 3.4- and 3.7- fold increases for AZD1775/Vorinostat or /SBHA, respectively), consistent with premature mitotic entry at this early interval. Interestingly, AZD1775/HDACI co-treatment for 16 h sharply arrested cells in early S-phase and increased newly replicated DNA incorporating EdU, accompanied by persistent increases in premature mitotic entry (e.g., increased p-H3 MI). Fluorescence microscopy demonstrated robust increases in both p-H3- and EdU-positive cells after 16-h co-exposure to AZD1775 and HDACIs. Furthermore, confocal microscopy of AZD1775/HDACI-treated cells (16 h) revealed markedly aberrant mitosis characterized by multiple mitotic abnormalities e.g., anaphase bridging, mono- or multi-polar spindles, centrosome clustering, etc. in p-H3-positive cells. These events were associated with a pronounced increase in DNA damage, manifested by expression of the double-strand break (DSB) indicator γH2A.X, as well as marked caspase activation and PARP cleavage in both p53-wt and -deficient cells. Similar results (e.g., cdc2/Cdk1 dephosphorylation of both Y15 and T14, γH2A.X expression, and PARP cleavage) were observed in a murine AML xenograft model in association with a significant reduction in tumor burden and prolongation of animal survival. In conclusion, these findings suggest that a novel strategy combining AZD1775 with HDACIs may markedly improve the anti-AML effectiveness of Wee1-targeting therapy, due to abrogation of both the G1/S and G2/M checkpoints through disruption of compensatory Chk1-mediated cdc2/Cdk1 T14 phosphorylation, and that this interaction occurs independently of p53 status. Disclosures No relevant conflicts of interest to declare.

Interactions between the Chk1 inhibitor UCN-01 and the farnesyltransferase inhibitor L744832 were examined in human leukemia cells. Combined exposure of U937 cells to subtoxic concentrations of UCN-01 and L744832 resulted in a dramatic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary acute myeloid leukemia (AML) blasts. Coadministration of L744832 blocked UCN-01-mediated phosphorylation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), leading to down-regulation of phospho-cyclic adenosine monophosphate responsive element-binding protein (phospho-CREB) and -p90RSK and activation of p34cdc2 and stress-activated protein kinase/ERK kinase/c-Jun N-terminal kinase (SEK/JNK). Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -glycogen synthase kinase-3 (-GSK-3), -p70S6K, -mammalian target of rapamycin (-mTOR), -forkhead transcription factor (-FKHR), -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis but did not prevent activation of p34cdc2 and JNK or inactivation of MEK/ERK and Akt. Enforced expression of myristoylated Akt but not constitutively active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01 and, in so doing, dramatically increase mitochondria-dependent apoptosis. (Blood. 2005;105:1706-1716)

The effects of combined exposure to the checkpoint abrogator UCN-01 and pharmacologic MEK1/2 inhibitors were examined in human multiple myeloma (MM) cell lines. Treatment of RPMI8226, NCI-H929, and U266 MM cells with a minimally toxic concentration of UCN-01 (150 nM) for 24 hours resulted in mitogen-activated protein (MAP) kinase activation, an effect that was blocked by coadministration of the MEK1/2 inhibitor PD184352. These events were accompanied by enhanced activation of p34cdc2 and a marked increase in mitochondrial damage (loss of ΔΨm; cytochrome c and Smac/DIABLO (direct IAP binding protein with low pI) release), poly(ADP-ribose) polymerase (PARP) cleavage, and apoptosis. PD184352/UCN-01 also dramatically reduced clonogenic survival in each of the MM cell lines. In contrast to As203, apoptosis induced by PD184352/UCN-01 was not blocked by the free-radical scavenger n-acetyl-l-cysteine. Whereas exogenous interleukin 6 substantially prevented dexamethasone-induced lethality in MM cells, it was unable to protect them from PD184352/UCN-01–induced apoptosis despite enhancing Akt activation. Insulinlike growth factor 1 (IGF-1) also failed to diminish apoptosis induced by this drug regimen. MM cell lines selected for a high degree of resistance to doxorubicin, melphalan, or dexamethasone, or displaying resistance secondary to fibronectin-mediated adherence, remained fully sensitive to PD184352/UCN-01–induced cell death. Finally, primary CD138+ MM cells were also susceptible to UCN-01/MEK inhibitor-mediated apoptosis. Together, these findings suggest that simultaneous disruption of cell cycle and MEK/MAP kinase signaling pathways provides a potent stimulus for mitochondrial damage and apoptosis in MM cells, and also indicate that this strategy bypasses the block to cell death conferred by several other well-described resistance mechanisms.

In diverse human cancers, oncogene-driven pathways antagonize the lethal effects of Bim, a major apoptosis executioner implicated in the activity of various anti-cancer agents including bortezomib (btz). As loss of Bim plays an important role in tumor development and growth as well as in acquisition of drug resistance, Bim represents an attractive therapeutic target, particularly for efforts to circumvent resistance to standard chemotherapy and diverse novel therapies. In the present study, the role of Bim in acquired resistance of multiple myeloma (MM) cells to btz was investigated. Bcl-2 family member expression profiles revealed high basal levels of Bim in most MM cell lines (Bimhi) and in primary CD138+ MM samples. However, Bimlow cells (e.g., H929) were highly susceptible to btz, arguing against the possibility that endogenous Bim levels correlate with btz sensitivity. In sharp contrast, shRNA Bim knockdown in Bimhi cells conferred pronounced resistance to btz. Interestingly, btz-resistant cells (PS-R) generated by exposure of U266 cells (Bimhi) to progressively increasing btz concentrations (to 20 nM) displayed markedly diminished Bim expression, a phenomenon validated in btz-resistant cells derived from other Bimhi cell lines (e.g., OPM2, RPMI8226). Notably, Bim up-regulation by histone deacetylase inhibitors (HDACIs e.g., SBHA), when combined with BH3-mimetics (e.g., ABT-737) that release Bim from binding to Bcl-2/Bcl-xL, effectively killed btz-resistant MM cells. Moreover, this strategy was highly active against primary CD138+ cells isolated from relapsed MM patients. In view of recent evidence that Bim negatively regulates autophagy (Luo et al., Mol Cell 47:359-70, 2012), the possibility that autophagy might also play a role in Bim-targeting strategies was investigated. Whereas drug-naïve and btz-resistant cells exhibited little difference in expression of autophagy-inducing proteins or autophagy initiation (e.g., by thapsigargin or tunicamycin), resistant cells displayed deficiencies in autophagosome maturation, reflected by delayed removal of GFP-LC3 puncta. Interestingly, SBHA co-administration largely abrogated ABT-737-induced autophagy and markedly increased the association between Beclin-1 and Bcl-2, accompanied by diminished binding of Bim to Bcl-2. Significantly, shRNA Bim knockdown strikingly increased autophagy in Bimhi cells. Moreover, SBHA was unable to inhibit autophagy in such cells due to failure of Bim up-regulation, resulting in a pronounced attenuation of SBHA/ABT-737-induced apoptosis. In Bimlow H929 cells, SBHA also failed to up-regulate Bim and potentiate ABT-737 lethality. However, disruption of autophagy by chloroquine (CQ) dramatically restored the sensitivity of these cells to this Bim-targeting regimen, an event associated with a striking increase in Bim expression. In contrast, CQ only modestly increased lethality in Bimhi cells in which autophagy was already disabled by SBHA-mediated Bim up-regulation. Moreover, compared to parental drug-naïve cells (Bimhi), btz-resistant cells displaying acquired loss of Bim were significantly more susceptible to potentiation of SBHA/ABT-737 lethality by CQ (e.g., 50% vs 22% increase in apoptosis; P < 0.01). Finally, in bim-/- MEFs derived from gene knockout mice, SBHA/ABT-737 co-treatment, with or without the addition of CQ, was unable to induce apoptosis, arguing that the presence of the bim gene is required for restoration of drug sensitivity by Bim-targeting therapy. Together, these findings argue that Bim deficiency represents a novel mechanism of adaptive (rather than intrinsic) btz-resistance in MM cells, and that simultaneous up-regulation of Bim (e.g., by HDACIs) and unleashing of Bim from binding to anti-apoptotic Bcl-2 family members (e.g., by BH3-mimetics such as ABT-737) can circumvent this form of resistance. They also raise the possibility that in addition to direct induction of apoptosis, Bim-mediated suppression of autophagy contributes to the lethality of Bim-targeting strategies, and that disruption of the latter process (e.g., by CQ) may be particularly beneficial in cells in which Bim up-regulation is disabled. Collectively, these findings suggest that Bim-targeting therapy may represent a novel and effective strategy in overcoming btz resistance in MM. This study was supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

Abstract 1658 There is great interest in repurposing drugs currently available in the pharmacopeia to other indications like cancer as such an approach would significantly shorten the time needed and cost encumbered to provide new and effective cancer therapeutics. One such drug is the gold compound Auranofin (AF), which is FDA approved as a treatment for rheumatoid arthritis (RA). We demonstrate that AF induces MCL cell death with an LD50 of 1000 nM, 500 nM and 90 nM for Granta and Jeko cell lines, and cells derived from a patient biopsy, respectively. The increased sensitivity of primary MCL patient specimens to AF is confirmed on 4 additional patient samples tested. AF similarly induced DLBCL cell death with an LD50 of 500 nM, 500 nM and 1000 nM for OCI Ly-10, SUDHL-6 and −4 cell lines, respectively. Exposure of Granta cells to an AF concentration that induced cell death resulted in the generation of reactive oxygen species (ROS). Pre-treatment of these cells with N-acetyl-cysteine (NAC) or glutathione (GSH) abrogated both ROS generation and the induction of cell death supporting the notion that AF induces NHL cell death through a redox dependent pathway. Although AF does increase mitochondrial membrane permeability (although not through the classical permeability transition pore), the major mechanism through which AF induces NHL cell death is activation of the extrinsic apoptotic pathway. In this regard, AF induces the activation of caspases 7 and 8 and results in PARP cleavage, all of which are blocked by NAC. Despite the fact that AF is a known inhibitor of thioredoxin reductase (TR), its cytotoxic effect is independent of TR inhibition, as we observe no difference in the response to AF in U266 multiple myeloma cells transfected with an expression vector which results in the over-expression of TR. Given the redox dependence of AF-induced cytotoxicity we hypothesized that inhibition of the glutathione system with buthionine sulphoximine (BSO) would further augment AF induced cell death. To test this hypothesis, Granta cells were exposed to both AF and BSO. Significant synergistic interactions of these drugs were seen when tested in a Laska analysis of synergy. For example, whereas the LD50 for AF alone in Granta cells was 1000 nM, the LD50 for AF in combination with 5 μM BSO was 200nm; for Ly-10 cells, the LD50 for AF±BSO was 400 nm vs. 190 nM, respectively, and for SUDHL-6, 411 nM vs. 185 nM, respectively. Similar results were seen in MCL cells flow cytometrically sorted from patient biopsy specimens. As observed in studies using AF alone, the cytotoxic effects of the combination were blocked with both NAC and GSH. Similar to results with AF alone, the synergistic effects of AF and BSO on NHL cytotoxicity were independent of their effects on TR. Exposure of Granta cells to AF resulted in NF-κB inhibition. NF-κB was further inhibited with concomitant exposure to BSO over-expression of relA in Granta cells, however, had no effect on AF and BSO induced cell death, suggesting the synergistic effects of AF and BSO on NHL cell death may be NF-κB-independent. Finally, we demonstrate that AF and BSO modify free thiols on the plasma membrane. As we have recently shown that the redox agent parthenolide induces NHL death in part by modifying surface protein thiols, AF and BSO may induce NHL cell death through a similar mechanism. In summary, AF induces both MCL and DLCL cell death through a redox-dependent pathway that involves the extrinsic apoptotic pathway. Profound synergy is seen with concomitant depletion of glutathione by BSO. The data presented above, along with the fact that AF is a drug having a favorable safety profile in patients, and is an FDA-approved drug for the treatment of RA makes it an attractive candidate for further study as a single agent and in rational combination with other redox active drugs. Disclosures: No relevant conflicts of interest to declare.

Abstract 3266 Previous studies have demonstrated interactions between histone deacetylase (HDAC) and proteasome inhibitors (PIs) in multiple myeloma, NHL, and CLL. However, exploration of this strategy in acute leukemias has been more limited. In this context, we have previously demonstrated that HDACIs activate the cytoprotective NF-κB pathway in acute myeloid leukemia (AML) cells, and that interruption of this process dramatically increases lethality. Such findings raise the possibility that PIs, which block degradation of the NF-κB-inhibitory protein IκBα, may act via an analogous mechanism in acute leukemias. Consequently, interactions between the clinically relevant pan-HDAC inhibitor belinostat (PXD-101) and the FDA-approved proteasome inhibitor bortezomib were evaluated in both continuously cultured cell lines and primary AML and acute lymphoid leukemia (ALL) samples. First, whereas each agent individually displayed only modest toxicity, co-treatment for 24 hr or 48 hr with low concentrations of bortezomib (3 - 5 nM) and belinostat (50 - 300 nM) led to pronounced increases in apoptosis in diverse human acute leukemia cell lines (e.g., AML, U937, HL-60, MV-4-11/Flt3-ITD; T-cell ALL, Jurkat; B-cell ALL, SEM). Interactions between these agents were determined to be synergistic by Median Dose Effect analysis. Significantly, equivalent interactions were observed in multiple primary AML (n = 4) and ALL (n = 3) blast specimens, while largely sparing normal CD34+ hematopoietic cells isolated from umbilical cord blood (n = 4), as determined by annexin V/PI, DiOC6, and/or 7-AAD uptake by flow cytometry. Western blot analysis demonstrated that co-exposure of primary leukemia blasts to bortezomib and belinostat resulted in marked increase in PARP cleavage, compared with each agent administrated alone. In addition, cell morphology exhibited classical features of apoptosis in primary acute leukemia blasts, but not in normal CD34+ cells, following combination treatment. Second, in both cell lines and primary blasts, administration of bortezomib resulted in accumulation of the phosphorylated (S32/S36) form of IκBα, accompanied by diminished belinostat-mediated hyperacetylation (K310) of RelA/p65. Bortezomib also blocked processing of the precursor p100 into the active p52, an event enhanced by co-treatment with belinostat. These results indicate that a regimen combining bortezomib and belinostat interrupts both canonical and non-canonical NF-κB signaling pathways in acute leukemia cells. Moreover, co-exposure to these agents diminished expression of NF-κB-dependent pro-survival proteins including Bcl-xL, XIAP, and SOD2, but not NF-κB-independent anti-apoptotic proteins such as survivin. Third, because the BH3-only Bcl-2 family pro-apoptotic protein Bim plays an important role in the lethality of PIs or HDACIs as single agents, the expression and functional role of Bim in bortezomib/belinostat interactions was examined. Notably, whereas treatment with marginally toxic concentrations of either agent alone clearly increased Bim protein levels, co-exposure of either leukemia cell lines or primary blasts to bortezomib and belinostat led to sharply increased Bim expression (particularly the BimEL isoform). Importantly, shRNA knock-down of Bim substantially attenuated lethality mediated by co-treatment with bortezomib and belinostat in both AML (U937) and ALL (Jurkat) cells, supporting the notion that up-regulation of Bim plays a critical role in anti-leukemic activity of the combination regimen. Lastly, exposure of cultured leukemia cells and primary blasts to belinostat ± bortezomib induced hyperacetylation of a-tubulin, indicating inhibition of HDAC6, a microtubule-associated deacetylase that regulates aggresome formation and cell survival in response to misfolded protein-induced stress. Together, these findings indicate that the regimen combining belinostat and bortezomib is highly active against human AML and ALL cells, including primary leukemic blasts, in association with perturbation in the balance between pro-survival (NF-κB-dependent) and pro-death (e.g., Bim) signals. They also suggest that this strategy warrants further attention in acute leukemias. Accordingly, a Phase I trial of belinostat and bortezomib in patients with refractory acute leukemia or MDS has recently been initiated. Disclosures: Off Label Use: Investigational use of belinostat and bortezomib.

Abstract 297 Background: High expression of the anti-apoptotic Bcl-2 family protein Mcl-1 is a critical survival mechanism in human multiple myeloma (MM) cells, and a key barrier to apoptosis induced by the Bcl-2/-xL antagonist ABT-737. De novo expression of the short-lived protein Mcl-1 is controlled transcriptionally by CDK9, a catalytic subunit of the PTEF-b transcription elongation complex (CDK9/cyclin T) that phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) to promote elongation of nascent transcripts. Thus, approaches targeting CDK9 may enhance ABT-737 efficacy in MM cells. Methods: To test this hypothesis, we employed the pan-CDK inhibitor roscovitine and a highly-selective CDK9 inhibitor (CDK9i) in drug-naïve and drug-resistant MM cell lines, as well primary plasma cells. Results: Co-administration of non- or sub-toxic concentrations of roscovitine or CDK9i interacted in a highly synergistic manner with ABT-737 (e.g., 150-300 nM) to induce apoptosis in U266, RPMI 8226, H929, OPM-2, and MM.1S MM cells, as well as IL-6-depedent ANBL-6 and KAS 6/1 cells. In each case, Combination Index values were significantly less than 1.0. These regimens were fully active in various drug-resistant MM cells, including U266/VR and OPM-2/VR bortezomib-resistant cells, RPMI 8226/RR lenalidomide-resistant cells, MM.1R dexamethasone-resistant cells, RPMI 8226/LR5 melphalan-resistant cells, and RPMI 8226/Dox40 doxorubicin-resistant cells. They also effectively killed MM cells in the presence of stromal cells or growth factors (i.e., IL-6, IGF-1, BAFF, and APRIL). Co-administration of roscovitine significantly enhanced ABT-737 lethality in primary CD138+ MM cells, while largely sparing CD138− bone marrow cells. Notably, the majority of MM cell lines exhibited high expression of cyclin T1 rather than cyclin T2a/b, in association with constitutively-activated CDK9, manifested by high levels of phosphorylation of CDK9 and RNA pol II CTD at Ser2 and 5. Exposure of MM cells to either roscovitine or CDK9i strikingly reduced RNA pol II CTD phosphorylation at both sites (particularly Ser5), but did not affect CDK9 and cyclin T1 levels. Whereas treatment with ABT-737 alone led to a modest but clear increase in Mcl-1 expression, roscovitine or CDK9i completely blocked this event and substantially down-regulated basal Mcl-1 expression. Notably, similar phenomena occurred in U266/VR cells, which displayed higher basal levels of Mcl-1, an important candidate resistance mechanism, compared to parental cells. Moreover, knock down of Mcl-1 by stable transfection with shRNA dramatically increased U266 sensitivity to ABT-737. Co-treatment of MM cells with ABT-737 and roscovitine or CDK9i did not affect expression of other anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL, but induced a Bcl-2 cleavage fragment. Expression profiling of BH3-only proteins in MM cells co-exposed to roscovitine and ABT-737 revealed a modest or a marked increase in expression of Bim (both EL and L isoforms) and Noxa (particularly a ∼27 kDa species), respectively, accompanied by a clear decrease in Puma levels. No changes in expression of other BH-3-only proteins (e.g., Bid, Bad, Bik, BMF, Hrk) were observed. Interestingly, knock down of Bim by stable transfection with shRNA dramatically diminished roscovitine/ABT-737 lethality, whereas Noxa knock-down by shRNA only partially protected cells from this regimen. In contrast, both shRNA approaches substantially protected U266 cells from bortezomib lethality. Conclusions: Collectively, these findings provide a mechanistic framework for targeting transcription by CDK9 inhibitors to diminish ABT-737-mediated Mcl-1 upregulation and to downregulate basal Mcl-1 levels, resulting in highly synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents. They also suggest that the BH3-only protein Bim may be required for apoptosis triggered by this strategy, while up-regulation of Noxa may amplify MM cell killing in this setting. Disclosures: No relevant conflicts of interest to declare.

MK-0457/VX-680 was developed as a pan-aurora kinase inhibitor that induces apoptosis in tumor cells and suppresses tumor growth in vivo. Recently, it was found that MK-0457 is also a potent inhibitor of both wild type and mutated Bcr/Abl kinases, including the T315I mutant which confers resistance to first- (i.e., imatinib mesylate/IM) and second-generation kinase inhibitors (e.g., dasatinib and nilotinib). Moreover, MK-0457 exhibits significant activity in patients with T315I phenotype-refractory CML or Ph-positive ALL. Here we examined interactions between MK-0457 and the histone deacetylase inhibitor (HDACI) vorinostat/SAHA in Bcr/Abl+ leukemia cells, including those resistant to IM through various mechanisms, particularly the T315I mutation. Co-administration of non- or subtoxic concentrations of vorinostat (0.5–2 μM) increased the lethality of MK-0457 (5–100 nM) in a highly synergistic manner in K562 and LAMA84 cells. Similar interactions were observed in primary CD34+ CML cells, whereas the combination largely spared normal bone marrow mononuclear cells. Moreover, the MK-0457/vorinostat regimen markedly induced cell death in IM-resistant K562 cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, a phenomenon accompanied by Lyn inactivation. Notably, the regimen was highly active against BaF/3 cells bearing various Bcr/Abl point mutations (i.e., T315I, E255K, and M351T) conferring resistance to IM. These events were associated with inactivation and downregulation of wild type and mutated Bcr/Abl (particularly T315I), reduced expression of phospho-CrkL, and vorinostat-mediated interruption of interactions between Bcr/Abl and Hsp90. Moreover, exposure of cells to marginally toxic doses of MK-0457 resulted in accumulation of cells with greater than 4N DNA content, resulting from induction of endoreduplication. Co-administration of vorinostat strikingly enhanced aurora kinase inhibition by MK-0457, reflected by markedly diminished expression of phospho-histone H3 (Ser 10), accompanied by preferential killing of polyploid cells. Interestingly, vorinostat also interacted with a selective inhibitor of aurora kinase A and B, at concentrations that inhibited histone H3 phosphorylation, to potentiate apoptosis in CML cells without modifying expression of phospho-Bcr/Abl or -CrkL. These results suggest that aurora kinase inhibition plays a functional role in synergistic interactions between HDACI and aurora kinase inhibitors. Finally, vorinostat markedly induced expression of the pro-apoptotic BH3-only protein Bim in various CML cell lines, including BaF/3 cells expressing the T315I Bcr/Abl mutation, as well as in primary CML cells. Knockdown of Bim by siRNA significantly protected K562 cells from apoptosis induced by vorinostat but not MK-0457. Furthermore, siRNA-mediated Bim downregulation blocked induction of Bim expression by vorinostat and dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. In addition, blockade of apoptosis by Bim siRNA did not modify downregulation of Bcr/Abl or phospho-histone H3 in cells exposed to the vorinostat/MK-0457 regimen, arguing against the possibility that the latter phenomena are secondary to activation of apoptotic cascade. Together, these findings indicate that vorinostat dramatically increases MK-0457 lethality in IM-sensitive and -resistant CML cells, and suggest that inactivation of Bcr/Abl kinase and aurora kinases, as well as Bim induction by vorinostat, contribute to this phenomenon.

Ras mutations, which result in constitutive Ras activation, occur frequently in human malignancies, including leukemia. This finding has prompted the development of farnesyltransferase inhibitors (FTIs), which interfere with Ras farnesylation and membrane translocation necessary for Ras function. However, FTIs alone have not yet fulfilled their clinical potential, raising the possibility that their role may lie in combination with other agents. The present studies examined interactions between the farnesyltransferase inhibitor L744832 and the Chk1 inhibitor UCN-01 in human leukemia cells. Combined exposure of U937 cells to sub-toxic concentrations of UCN-01 (100nM) and the FTI L744352 (10μM) resulted in a dramatic and highly synergistic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary AML blasts, and with other farnesyltransferase inhibitors (e.g., FTI-277). These events were accompanied by cleavage of the anti-apoptotic proteins Bcl-2, XIAP, and Mcl-1. Co-administration of L744832 blocked UCN-01-mediated phosphorylation of MEK/ERK, leading to down-regulation of phospho-CREB and -p90RSK, and activation of p34cdc2 and SEK/JNK. Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -GSK, -p70S6K, -mTOR, -FKHR, -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis, but did not prevent activation of p34cdc2 and JNK, or inactivation of MEK/ERK and Akt. Enforced expression of myristolated Akt but not constitutively-active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with constitutively active Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01, and in so doing, dramatically increase mitochondria-dependent apoptosis. They also raise the possibility that combined treatment with FTIs and UCN-01 may represent a novel therapeutic strategy in leukemia.