Bortezomib Resistance Research Articles

Inhibition of thioredoxin activates mitophagy and overcomes adaptive bortezomib resistance in multiple myeloma

BackgroundAlthough current chemotherapy using bortezomib (Velcade) against multiple myeloma in adults has achieved significant responses and even remission, a majority of patients will develop acquired resistance to bortezomib. Increased thioredoxin level has been reported to be associated with carcinogenesis; however, the role of thioredoxin in bortezomib drug resistance of myeloma remains unclear.MethodsWe generated several bortezomib-resistant myeloma cell lines by serially passaging with increased concentrations of bortezomib over a period of 1.5 years. Thioredoxin expression was measured by real-time PCR and western blot.ResultsThe role of thioredoxin in the survival of bortezomib-resistant myeloma cells was determined by specific shRNA knockdown in vitro and in vivo. Thioredoxin inhibitor (PX12) was used to determine the effectiveness of thioredoxin inhibition in the treatment of bortezomib-resistant myeloma cells. The effect of thioredoxin inhibition on mitophagy induction was examined. The correlation of thioredoxin expression with patient overall survival was interrogated. Thioredoxin expression was significantly upregulated in bortezomib-resistant myeloma cells and the change correlated with the increase of bortezomib concentration. Thioredoxin gene knockdown using specific shRNA sensitized bortezomib-resistant myeloma cells to bortezomib efficiency in vitro and in vivo. Similarly, pharmacological inhibition with PX12 inhibited the growth of bortezomib-resistant myeloma cells and overcame bortezomib resistance in vitro and in vivo. Furthermore, inhibition of thioredoxin resulted in the activation of mitophagy and blockage of mitophagy prevented the effects of PX12 on bortezomib-resistant myeloma cells, indicating that mitophagy is the important molecular mechanism for the induction of cell death in bortezomib-resistant myeloma cells by PX12. Moreover, inhibition of thioredoxin resulted in downregulation of phosphorylated mTOR and ERK1/2. Finally, thioredoxin was overexpressed in primary myeloma cells isolated from bortezomib-resistant myeloma patients and overexpression of thioredoxin correlated with poor overall survival in patients with multiple myeloma.ConclusionsOur findings demonstrated that increased thioredoxin plays a critical role in bortezomib resistance in multiple myeloma through mitophagy inactivation and increased mTOR and ERK1/2 phosphorylation. Thioredoxin provides a potential target for clinical therapeutics against multiple myeloma, particularly for bortezomib-resistant/refractory myeloma patients.

Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

SummaryBackground The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Pirh2 mediates the sensitivity of myeloma cells to bortezomib via canonical NF-\u03baB signaling pathway

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factor-kappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

Nuclear factor-κB (NF-κB) is a family of transcription factors that play a key role in cell survival and proliferation in many hematological malignancies, including multiple myeloma (MM). Bortezomib, a proteasome inhibitor used in the management of MM, can inhibit both canonical and noncanonical activation of NF-κB in MM cells. However, we previously reported that a significant fraction of freshly isolated MM cells harbor bortezomib-resistant NF-κB activity. Here, we report that hyaluronan and proteoglycan link protein 1 (HAPLN1) is produced in bone marrow stromal cells from MM patients, is detected in patients' bone marrow plasma, and can activate an atypical bortezomib-resistant NF-κB pathway in MM cells. We found that this pathway involves bortezomib-resistant degradation of the inhibitor of NF-κB (IκBα), despite efficient bortezomib-mediated inhibition of proteasome activity. Moreover, HAPLN1 can also confer bortezomib-resistant survival of MM cells. We propose that HAPLN1 is a novel pathogenic factor in MM that induces an atypical NF-κB activation and thereby promotes bortezomib resistance in MM cells.

Inhibition of O-GlcNAcase Sensitizes Apoptosis and Reverses Bortezomib Resistance in Mantle Cell Lymphoma through Modification of Truncated Bid.

Aberrant energy metabolism represents a hallmark of cancer and contributes to numerous aggressive behaviors of cancer cells, including cell death and survival. Despite the poor prognosis of mantle cell lymphoma (MCL), due to the inevitable development of drug resistance, metabolic reprograming of MCL cells remains an unexplored area. Posttranslational modification of proteins via O-GlcNAcylation is an ideal sensor for nutritional changes mediated by O-GlcNAc transferase (OGT) and is removed by O-GlcNAcase (OGA). Using various small-molecule inhibitors of OGT and OGA, we found for the first time that O-GlcNAcylation potentiates MCL response to bortezomib. CRISPR interference of MGEA5 (encoding OGA) validated the apoptosis sensitization by O-GlcNAcylation and OGA inhibition. To identify the potential clinical candidates, we tested MCL response to drug-like OGA inhibitor, ketoconazole, and verified that it exerts similar sensitizing effect on bortezomib-induced apoptosis. Investigations into the underlying molecular mechanisms reveal that bortezomib and ketoconazole act in concert to cause the accumulation of truncated Bid (tBid). Not only does ketoconazole potentiate tBid induction, but also increases tBid stability through O-GlcNAcylation that interferes with tBid ubiquitination and proteasomal degradation. Remarkably, ketoconazole strongly enhances bortezomib-induced apoptosis in de novo bortezomib-resistant MCL cells and in patient-derived primary cells with minimal cytotoxic effect on normal peripheral blood mononuclear cells and hepatocytes, suggesting its potential utility as a safe and effective adjuvant for MCL. Together, our findings provide novel evidence that combination of bortezomib and ketoconazole or other OGA inhibitors may present a promising strategy for the treatment of drug-resistant MCL. Mol Cancer Ther; 17(2); 484-96. ©2017 AACR.

Unbiased compound-protein interface mapping and prediction of chemoresistance loci through forward genetics in haploid stem cells.

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis.Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca2+ pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes.Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces.

Abstract A136: KDM5A activity inhibition by 2-PCPA: overcoming tolerance to bortezomib

Abstract Purpose: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for myeloma treatment. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance remain to be understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To overcome bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant and bortezomib-sensitive IM-9 cell lines in response to bortezomib, and expressions of KDM5A genes were significantly increased. Methods: The differential gene expression profiles of bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cell lines in response to bortezomib were performed using Affymetrix GeneChip. To confirm the results, real-time PCR and Western blot analysis were performed. Bortezomib-resistant IM-9 cells were treated with or without bortezomib and</>or histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride (2-PCPA). Moreover, level of histone methylation was examined by Western blot analysis. Results: At concentrations that effectively inhibited proteasome activity (maximum dose with 100nM), bortezomib induced cell death in bortezomib-sensitive IM-9 cells, but not in bortezomib-resistant IM-9. In comparison of differential gene expression profiles between bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cells, we showed overexpression of KDM5A, which is associated with chromatin-mediated reversible drug-tolerant state, Moreover, 2-PCPA overcomes bortezomib resistance in a dose-dependent manner. The histone demethylation at H3K4, H3K9, and H3K27 was suppressed with 2-PCPA. Conclusion: Histone lysine-specific demethylase inhibitor, 2-PCPA, with bortezomib may be useful for overcoming bortezomib resistance in myeloma cells by suppression of KDM3A and KDM5A activities. Citation Format: Yasuhito Terui, Yuji Mishima, Kiyohiko Hatake. KDM5A activity inhibition by 2-PCPA: overcoming tolerance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A136.

Endoplasmic reticulum stress in the development of multiple myeloma and drug resistance.

Multiple myeloma (MM) is a haematological malignancy of mature antibody‐secreting plasma cells. Currently, MM is incurable, but advances in drug treatments have increased patient lifespan. One of the characteristics of MM is the excessive production of monoclonal immunoglobulin (also referred to as paraprotein). This high level of protein production induces endoplasmic reticulum (ER) stress, and proteasomal degradation of the paraprotein is required to avoid ER stress‐induced cell death. Consequently, proteasomal inhibitors such as bortezomib have been particularly effective therapies. Unfortunately development of resistance to bortezomib is common. In this review, we address how control of endoplasmic reticulum stress is important in the development of MM and how the unfolded protein response and its associated stress response pathways are involved in the development of bortezomib resistance.

Truncated protein tyrosine phosphatase receptor type O suppresses AKT signaling through IQ motif containing GTPase activating protein 1 and confers sensitivity to bortezomib in multiple myeloma.

Proteasome inhibitors are an important part of our chemotherapeutic armamentarium against multiple myeloma, but the vast majority of patients eventually develop drug-resistant disease through incompletely understood mechanisms. Comparison of gene expression profiles (GEPs) of bortezomib-resistant (BR) myeloma cell lines with their drug-naïve counterparts revealed decreased expression of truncated Protein tyrosine phosphatase receptor-type O (PTPROt) in BR cells. Over-expression of wild-type PTPROt in drug-naïve and BR cells reduced myeloma cell proliferation, induced apoptosis, and sensitized cells to bortezomib and to alkylating agents. PTPROt expression reduced AKT phosphorylation and activity, and sensitized to pharmacologic AKT pathway inhibitors, but this was not the case for a substrate-trapping catalytic domain-inactivating mutant. Co-immunoprecipitation and mass spectrometry studies identified IQ motif containing GTPase activating protein 1 (IQGAP1) as a PTPROt binding partner, and PTPROt reduced tyrosine phosphorylation of IQGAP1, providing a link to AKT activity. Analysis of clinically annotated GEP databases identified high PTPROt expression as being related to an increased likelihood of achieving complete remission with bortezomib therapy, while low expression was linked to a greater likelihood of disease progression. Finally, high PTPROt expression associated with prolonged median overall survival in patients receiving bortezomib-based therapy in the front-line or relapsed and/or refractory settings. Taken together, these data identify PTPROt suppression as a novel mechanism of myeloma resistance to bortezomib in myeloma cell lines, and also support the possibility that PTPROt expression could be used as a biomarker to predict outcomes with bortezomib, and by which to select patients for therapy with AKT inhibitors.

YM155 exerts potent cytotoxic activity against quiescent (G0/G1) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1.

YM155, a novel small molecule inhibitor of survivin, shows broad anticancer activity. Here, we have focused on the cytotoxic activity of YM155 against multiple myeloma (MM) including cytokinetically quiescent (G0/G1) cells and bortezomib resistant cells. YM155 strongly inhibited the growth of MM cell lines with the IC50 value of below 10 nM. YM155 also showed potent anti-myeloma activity in mouse xenograft model. YM155 suppressed the expression of survivin and rapidly directed Mcl-1 protein for proteasome degradation. YM155 abrogated the interleukin-6-induced STAT3 phosphorylation, subsequently blocked Mcl-1 expression and induced apoptosis in MM cells. Triple-color flow cytometric analysis revealed that YM155 potently induced cell death of MM cells in G0 phase. Quiescent primary MM cells were also sensitive to YM155. We established bortezomib-resistant MM cell line, U266/BTZR1, which possess a point mutation G322A. YM155 exhibited similar cytotoxic potency against U266/BTZR1 compared with parental cells. Interestingly, survivin expression was markedly elevated in U266/BTZR1 cells. Treatment with YM155 significantly down-regulated this increased survivin and Mcl-1 expression in U266/BTZR1 cells. In conclusion, our data indicate that YM155 exhibits potent cytotoxicity against quiescent (G0/G1) MM cells and bortezomib-resistant cells. These unique features of YM155 may be beneficial for the development of new therapeutic strategies to eliminate quiescent MM cells and overcome bortezomib resistance.

CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma.

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862-70. ©2017 AACR.

Establishment of BOR-Resistant U266 Cell Line and the Detection of Its Biological Activities

To establish bortezomib (BOR)-resistant human multiple myeloma U266 cell line U266/BOR and to detect its biological characteristics. U266 cells were constantly exposed at low dose and progressively increasing dose of BOR to establish U266/BOR, the cell morphology was observed by inverted microscopy, IC50 and resistant index were determined by MTT assay, cell growth curve was drawed and the doubling time was calculated; cell cycle distribution were analyzed by flow cytometry, and RT-PCR was used to detect the mRNA expression of resistance-related genes. The MM U266/BOR cell line was successfully constructed and its resistance index was up to 19.8. The both cell morphologies were not different. Compared with U266 cells, the multiplication time was postponed with the increase of G0/G1 cell ratio, and S phase was reduced. The mRNA expression of PTPROt, Beclin 1 and PTEN were reduced, and the mRNA expression of c-Maf was enhanced in U266/BOR cells; as compared with U266 cells, but the MDR1 mRNA expression was not different between U266 cells and U266 BOR cells. The BOR-resistant U266 cell line has been establiseed successfully. It provides an ideal cell model for further exploration of the mechanism for BOR resistance.

BAFF is involved in macrophage-induced bortezomib resistance in myeloma

We aimed to characterize the role of B-cell activating factor (BAFF) in macrophage-mediated resistance of multiple myeloma (MM) cells to bortezomib (bort), and to further understand the molecular mechanisms involved in the process. First, we detected BAFF and its three receptors on myeloma cells and macrophages using the quantitative reverse transcriptase-polymerase chain reaction and flow cytometry. The secretion of BAFF was tested in patients with MM, MM cell lines, and macrophages. The ability of macrophages to protect MM cells from bort-induced apoptosis was significantly attenuated using BAFF-neutralizing antibody in the co-culture system or knocking down the expression of BAFF in macrophages with small interfering RNA. We also showed that the MM–macrophage interaction through BAFF and its receptors was primarily mediated by the activation of Src, Erk1/2, Akt, and nuclear factor kappa B signaling and the suppression of caspase activation induced by bort. Our data demonstrated that BAFF played a functional role in the macrophage-mediated resistance of MM cells to bort, suggesting that targeting BAFF may provide a basis for the molecular- and immune-targeted therapeutic approach.

Bortezomib resistance in multiple myeloma is associated with increased serine synthesis

BackgroundThe proteasome inhibitor bortezomib (BTZ) is successfully applied in the treatment of multiple myeloma, but its efficacy is restricted by the wide-spread occurrence of resistance. Metabolic alterations play an important role in cancer development and aid in the cellular adaptation to pharmacologically changed environments. Metabolic changes could therefore play an essential role in the development of drug resistance. However, specific metabolic pathways that can be targeted to improve bortezomib therapy remain unidentified.MethodsWe elucidated the metabolic mechanisms underlying bortezomib resistance by using mass spectrometry-based metabolomics and proteomics on BTZ-sensitive and BTZ–resistant multiple myeloma cell lines as well as in a set of CD138+ cells obtained from multiple myeloma patients.ResultsOur findings demonstrate that a rewired glucose metabolism sustains bortezomib resistance. Mechanistically, this results in higher activity of both the pentose phosphate pathway and serine synthesis pathway, ultimately leading to an increased anti-oxidant capacity of BTZ-resistant cells. Moreover, our results link both serine synthesis pathway activity and expression of 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of serine synthesis, to bortezomib resistance across different BTZ-resistant multiple myeloma cell lines. Consistently, serine starvation enhanced the cytotoxicity of bortezomib, underscoring the importance of serine metabolism in the response to BTZ. Importantly, in CD138+ cells of clinically bortezomib refractory multiple myeloma patients, PHGDH expression was also markedly increased.ConclusionsOur findings indicate that interfering with serine metabolism may be a novel strategy to improve bortezomib therapy and identify PHGDH as a potential biomarker for BTZ resistance.

Bifurcated BACH2 control coordinates mantle cell lymphoma survival and dispersal during hypoxia

AbstractBACH2, a B-cell–specific transcription factor, plays a critical role in oxidative stress–mediated drug resistance in mantle cell lymphoma (MCL); however, the biological functions of BACH2 and its regulation of B-cell malignancies in chronic hypoxic microenvironment have not been studied. Here, we found that silencing BACH2 led to not only increased tumor formation and colony formation but also increased tumor dispersal to spleen and bone marrow. Decreased BACH2 levels in patients were also correlated with bone marrow and gastrointestinal dispersal of MCL and blastoid subtypes of MCL. Unexpectedly, decreased BACH2 levels in dispersed MCL cells were due to direct transcriptional repression by hypoxia-induced factor 1α (HIF-1α) and increased heme-mediated protein degradation. In normoxic conditions, BACH2 was able to modulate HIF-1α degradation by suppressing prolyl hydroxylase 3 expression. Bifurcated BACH2 controls during hypoxia and normoxia coordinate not only MCL tumor dispersal but also drug resistance, including bortezomib resistance, via plasmacytic differentiation. Our data highlight an interactive relationship between tumor cells and local microenvironment and the mechanisms of B-cell transcription factor in the regulation of MCL dispersal.

Positive transcription elongation factor b (P-TEFb) is a therapeutic target in human multiple myeloma.

The role of the positive RNA Pol II regulator, P-TEFb (positive transcription elongation factor b), in maintenance of the anti-apoptotic protein Mcl-1 and bortezomib (btz) resistance was investigated in human multiple myeloma (MM) cells. Mcl-1 was up-regulated in all MM lines tested, including bortezomib-resistant lines, human MM xenograft mouse models, and primary CD138+ MM cells. Mcl-1 over-expression significantly reduced bortezomib lethality, indicating a functional role for Mcl-1 in bortezomib resistance. MM cell lines, primary MM specimens, and murine xenografts exhibited constitutive P-TEFb activation, manifested by high CTD (carboxy-terminal domain) S2 phosphorylation, associated with a) P-TEFb subunit up-regulation i.e., CDK9 (42 and 55 kDa isoforms) and cyclin T1; and b) marked CDK9 (42 kDa) T186 phosphorylation. In marked contrast, normal hematopoietic cells failed to exhibit up-regulation of p-CTD, CDK9, cyclin T1, or Mcl-1. CDK9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Moreover, CRISPR-Cas CDK9 knock-out triggered apoptosis in MM cells and dramatically diminished cell growth. Pan-CDK e.g., dinaciclib or alvocidib and selective CDK9 inhibitors (CDK9i) recapitulated the effects of genetic P-TEFb disruption. CDK9 shRNA or CDK9 inhibitors significantly potentiated the susceptibility of MM cells, including bortezomib-resistant cells, to proteasome inhibitors. Analogously, CDK9 or cyclin T1 knock-down or CDK9 inhibitors markedly increased BH3-mimetic lethality in bortezomib-resistant cells. Finally, pan-CDK inhibition reduced human drug-naïve or bortezomib-resistant CD138+ cells and restored bone marrow architecture in vivo. Collectively, these findings implicate constitutive P-TEFb activation in high Mcl-1 maintenance in MM, and validate targeting the P-TEFb complex to circumvent bortezomib-resistance.

Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells.

Purpose: The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma. Deubiquitylating enzyme USP1 participates in DNA damage response and cellular differentiation pathways. To date, the role of USP1 in multiple myeloma biology is not defined. In the present study, we investigated the functional significance of USP1 in multiple myeloma using genetic and biochemical approaches.Experimental Design: To investigate the role of USP1 in myeloma, we utilized USP1 inhibitor SJB3-019A (SJB) for studies in myeloma cell lines and patient multiple myeloma cells.Results: USP1-siRNA knockdown decreases multiple myeloma cell viability. USP1 inhibitor SJB selectively blocks USP1 enzymatic activity without blocking other DUBs. SJB also decreases the viability of multiple myeloma cell lines and patient tumor cells, inhibits bone marrow plasmacytoid dendritic cell-induced multiple myeloma cell growth, and overcomes bortezomib resistance. SJB triggers apoptosis in multiple myeloma cells via activation of caspase-3, caspase-8, and caspase-9. Moreover, SJB degrades USP1 and downstream inhibitor of DNA-binding proteins as well as inhibits DNA repair via blockade of Fanconi anemia pathway and homologous recombination. SJB also downregulates multiple myeloma stem cell renewal/survival-associated proteins Notch-1, Notch-2, SOX-4, and SOX-2. Moreover, SJB induced generation of more mature and differentiated plasma cells. Combination of SJB and HDACi ACY-1215, bortezomib, lenalidomide, or pomalidomide triggers synergistic cytotoxicity.Conclusions: Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel multiple myeloma therapy. Clin Cancer Res; 23(15); 4280-9. ©2017 AACR.

Abstract 2051: LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening

Abstract Large granular lymphocytic (LGL) leukemia is a rare form of leukemia that is caused by the increase in cytotoxic T-lymphocytes or natural killer (NK) cells. A subcategory of T-LGL leukemia that co-expresses T and NK cell surface markers is very aggressive and has a very poor prognosis. Current treatment of aggressive T-LGL is based on non-specific immunosuppressive therapy. Lack of model cell lines contributes to slow progress in designing targeted therapy and clinical trials. Our recent studies demonstrate that the mouse T-leukemic cell line LSTRA reproduces some important characteristics of aggressive T-LGL leukemia. This is the first report of a cell line model mimicking the deadly human leukemia and has great potentials for drug screening in treating T-LGL leukemia. Bortezomib is one of the drugs that show promising results in treating LGL leukemia patients in clinical trials. Bortezomib is the first FDA-approved reversible proteasome inhibitor in treating multiple myeloma and mantle cell lymphoma patients. One of the major concerns in using single-agent bortezomib is that patients develop resistance during the course of treatment and cancers relapse. Therefore, it is important to develop novel strategies in overcoming LGL leukemic cells’ resistance to bortezomib and its derivatives. In our current study, we showed that bortezomib inhibited cell proliferation and induced apoptosis in LSTRA leukemic cells. Partial reversion after 48-72 hr of bortezomib treatment suggested development of resistance to bortezomib. We further established bortezomib-resistant LSTRA cells that tolerated significantly higher levels of bortezomib. Both carfilzomib (FDA-approved second generation irreversible proteasome inhibitor) and MLN 4924 (proteasome pathway upstream inhibitor) failed to overcome bortezomib resistance. Only luteolin, a common flavonoid found in plants, can effectively overcome bortezomib resistance. Metabolic reprogramming has been linked to drug resistance in cancer cells. Indeed, We found that bortezomib-resistant LSTRA cells had smaller cell size as well as reduced rate of glucose uptake, lactate secretion, and mitochondrial respiration. These results suggest that reduced mitochondrial activity may be an important molecular marker for LSTRA cell’s resistance to bortezomib. All together, we identified luteolin as a potential small molecule in overcoming LGL leukemia’s resistance to proteasome inhibitor therapy. Altered mitochondrial activity and cell metabolism in bortezomib-resistant cells provide additional targets in developing novel combined chemotherapy for cancer patients. Citation Format: Fu-Shin Chueh, Fu-Yu Chueh, Chao-Lan Yu. LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2051. doi:10.1158/1538-7445.AM2017-2051

Abstract 1106: Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma

Abstract The proteasome inhibitor bortezomib is widely used in the treatment of patients with multiple myeloma (MM). The expression levels of many proteins increase as a result of bortezomib treatment, including the pro-apoptotic protein NOXA. NOXA functions to sequester the anti-apoptotic BCL-2 family member, MCL-1. High levels of MCL-1 and/or low levels of NOXA have been implicated in bortezomib resistance and negative patient outcomes, including short duration of treatment response. The BCL-2-specific BH3 mimetic venetoclax (ABT-199) has also been explored in multiple hematological malignancies, including the treatment of MM. Venetoclax induces apoptosis in a BCL-2 specific manner by directly inhibiting BCL-2 function. However, intrinsic resistance to venetoclax treatment observed in MM patient samples has been attributed to a low BCL-2-to-MCL-1 gene expression ratio, suggesting a central role for MCL-1 in cell survival in this context as well. Increased MCL-1 expression is a known resistance mechanism to venetoclax treatment in a variety of cell types including chronic lymphocytic leukemia and lymphomas. Considering the central role of MCL-1 to treatment efficacy in MM, we investigated the ability of an MCL-1-lowering agent, namely the CDK9 inhibitor alvocidib, to potentiate the activity of venetoclax in MM. Alvocidib suppresses MCL-1 expression via CDK9-mediated regulation of RNA polymerase II. Alvocidib has achieved robust improvements in the clinical response rates of high-risk, newly diagnosed acute myeloid leukemia (AML) patients as part of the time-sequential ACM regimen (alvocidib + cytarabine + mitoxantrone). We therefore hypothesized that alvocidib would potentiate the activity of venetoclax in MM through an MCL-1-dependent mechanism. In this report, we demonstrate that alvocidib inhibits the protein expression of MCL-1 in MM cells in a time-dependent fashion, up to 96 hours. In cell viability assays, the addition of up to 100 nM venetoclax resulted in a 2.8-fold reduction in the IC50 of alvocidib in the cultured OPM-2 cell line. Conversely, the potentiation of venetoclax activity with the addition of alvocidib resulted in a more than 500-fold decrease in IC50 in the relatively venetoclax-resistant OPM-2 cells. Additional studies are currently underway to investigate the efficacy of alvocidib and venetoclax in the context of bortezomib resistance where low NOXA may contribute to enhanced cell survival via MCL-1. Taken together, our data suggest that the combination of alvocidib with venetoclax may constitute a novel therapeutic regimen in the treatment of MM. Further, it suggests that CDK9-mediated targeting of MCL-1 may offer a clinical route to addressing intrinsic resistance in MM patients. Citation Format: Mark Livingston, Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2017-1106

Selinexor Overcomes Hypoxia-Induced Drug Resistance in Multiple Myeloma

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib.