Zhu, Yuan Xiao

Abstract Abstract 127 The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. A landmark paper has recently identified cereblon (CRBN) as a primary target of thalidomide teratogenicity. We hypothesized that this protein would also be required for myeloma (MM) cytotoxicity. To examine this issue we first tested 12 MM cell lines: 4 of which (OCIMY5, OPM1, SKMM2 and KMS12 PE) were very resistant to treatment with both lenalidomide and pomalidomide. Of these, OCIMY5 and OPM1 showed CRBN gene expression levels at the bottom 10% across the human MM cell lines. We next used CRBN shRNA lentiviral expression constructs to knock down CRBN in 5 human-derived myeloma cell lines (HMCLs) (KMS18, MM1.S, H929, OPM2 and JJN3). HMCLs infected with CRBN shRNAs have a significant reduction of CRBN expression and subsequent diminished cell viability (reduced 65–78%). Surviving MM cells with CRBN knock down demonstrate an acquired (essentially complete) resistance to lenalidomide when compared with controls but retained sensitivity to melphalan, dexamethasone and bortezomib. Gene expression changes induced by lenalidomide were dramatically suppressed in those CRBN depleted cells, e.g., OPM2 cells only showed 30 down regulated (3% of control) and 150 up-regulated genes (24% of control) after lenalidomide treatment, further emphasizing that CRBN is required for lenalidomide activity. We next compared by array comparative genomic hybridization the differences between MM1.S (lenalidomide sensitive) and its isogenic lenalidomide resistant line (MM1.S res), generated by culturing MM1.S in gradually increasing concentrations of lenalidomide. Only three structural abnormalities were noted differentiating the two lines one of which was deletion of CRBN. In fact the baseline MM1.S cell line has a minor population of 9% of cells with CRBN depletion which becomes dominant after lenalidomide treatment. However, further scrutiny of CRBN status in additional HMCLs and MM patients (irrespective of therapy and stage) suggested that copy-number abnormalities affecting this gene are uncommon events in MM, with 12% of HMCLs (7 out of 60) and only 1.2% of MM patients (3 out of 238) showing CRBN monoallelic deletion. Next, we examined by quantitative polymerase chain reaction (Q-PCR) ten MM patients who had become resistant to lenalidomide therapy. CRBN expression level by Q-PCR in 8 of 10 patient samples had a significant reduction (20% to 90% reduction compared to baseline) at time of drug resistance, suggesting CRBN expression as a potential biomarker to predict IMiD response. Notably however some clearly resistant patients have normal CRBN, demonstrating that while CRBN is absolutely required for response, multiple resistance mechanisms are likely present. By gene and protein expression downstream targets of CRBN were examined and included interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. In summary, we demonstrate here that CRBN is essential for IMiD activity and preliminary data support that low levels of CRBN predict for poor drug response. In addition, our data suggest that CRBN is a critical molecule but not the unique source of IMiD resistance. We suggest that the “IMiDs” be renamed Cereblon binding small molecules to more accurately reflect their mechanism of action. Disclosures: Orlowski: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

Abstract Abstract 1807 We have recently demonstrated that cereblon (CRBN) mediates the direct anti-myeloma activity of immunomodulatory drugs (IMiDs). However, the genes/pathways downstream of CRBN associated with anti-myeloma activity remain unclear. We, and others, identified interferon regulatory factor 4 (IRF4) as one of the downstream targets of CRBN-associated signaling. Both lenalidomide treatment and CRBN knockdown downregulate IRF4. IRF4 levels return to baseline in IMiD resistant cells surviving CRBN silencing. To determine whether IMiD-induced IRF4 downregulation is critical to anti-MM activity, we overexpressed IRF4 in two IMiD-sensitive human MM cell lines (HMCLs), KMS11 and MM1.S, followed by lenalidomide treatment. Lenalidomide-induced cytotoxicity was greatly impaired in both HMCLs overexpressing IRF4 compared with the control virus infected cells. Further analysis indicated that IRF4 over-expression does not completely prevent lenalidomide-induced growth arrest, but reduces cell death by 70% after lenalidomide treatment. Immunoblotting analysis of KMS11 cells indicated that IRF4 over-expression blocks lenalidomide-induced activation of caspase 8, reduces up-regulation of p21waf and increases CDK6 expression but does not significantly affect lenalidomide-induced MYC down-regulation. Although cereblon and IRF4 are broadly expressed in MM, baseline levels of expression are only weakly correlated (r=0.22) in primary MM patient gene expression analysis. Gene expression studies revealed statistical changes in 1,368 genes when comparing high versus low CRBN expression in primary myeloma samples. Interestingly genes associated with high CRBN expression included cyclin D2, SOCS3 and IL4 while genes associated with low cereblon expression included cyclin D1, FRZB and CD200. In order to understand how CRBN is connected with downstream anti-myeloma signaling, a structure-function study was performed to determine which CRBN domain is required for lenalidomide-induced IRF4 down-regulation and cytotoxicity. Lentiviral constructs expressing wild-type CRBN and a series of mutated CRBN were generated, including mutations at thalidomide binding site (Y384A/W386A), deletion of DDB1 binding region (ΔMid) and truncations at N-terminal and C-terminal. Lentiviruses from these constructs were used to infect IMiDs resistant HMCLs, OCI-MY5 and MM1.S res. Both of these cell lines have very low endogenous CRBN expression and they became sensitive to lenalidomide after introduction of wild-type CRBN. Conversely, introduction of CRBN with mutated thalidomide-binding site or with DDB1 binding region depletion failed to mediate lenalidomide toxicity and down-regulation of IRF4. OCI-MY5 cells expressing either N-terminal or C-terminal truncated CRBN showed substantial reduced responses (more than 50%) to lenalidomide compared with wild-type CRBN expressing cells. Deletion of only 20–30 amino acids at either ends of CRBN greatly impaired the protein function, suggesting that protein folding might be important for CRBN-mediated IMiD response. Our data indicate that IMiD induced myeloma cytotoxicity is largely mediated by modifying CRBN associated E3 ubiqutin ligase and subsequent IRF4 downregulation, suggesting the CRBN-IRF4 axis is a potential target for development of new anti-myeloma drugs. Disclosures: Schmidt: Karyopharm: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Abstract 573 Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease. Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures: Schmidt: Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Abstract 194 Background: Immunomodulatory drugs (IMiDs) are key components of treatment for hematologic malignancies, especially multiple myeloma (MM). Recently, we demonstrated that CRBN expression is the major mediator of IMiD action operating at least in part via interferon regulatory factor 4 (IRF4), however the clinical significance of this finding remains unclear. Methods: To better understand clinical impact, we retrospectively reviewed 148 MM patients whose tumor cells were tested for CRBN expression by Gene Expression Profiling (GEP) prior to treatment with an IMiD containing therapy. Results: GEP studies show no significant differences in CRBN expression between MGUS, smoldering MM (SMM), untreated symptomatic MM and normal plasma cells. However, differences were observed between MM cell lines of varying drug sensitivity to IMiDs and between other B-cell Neoplasia (eg low grade lymphoma has higher levels). Within the genetic subtypes of MM, levels were significantly higher for hyperdiploid patients (median 1.26) as opposed to significantly lower levels for TC class D2 (median 0.76) and average for the 4p16 (median 0.97). We next screened the University of Arkansas Medical School (UAMS) GEP database of MM patients treated with combination therapies and did not find significant correlation between CRBN expression and survival in this multi-agent regimen. We then focused on patients that received only single agent IMiD therapy with low dose dexamethasone. Review of the twelve lowest expressing patients demonstrated a lack of response to IMiDs in any patient. Finally we analyzed 53 relapsed or relapsed/refractory patients treated homogeneously in a study with pomalidomide 2–4mg daily and weekly dexamethasone 40mg. CRBN levels were assessed prior to therapy initiation. Response rates varied significantly based on gene expression level. The percentage of patients that demonstrated at least a partial response was 0% for CRBN <0.81, 19% for CRBN expression of 0.81–0.90, and 33% for CBN expression >0.9. Significant differences in PFS (3.0 months vs. 8.9 months, p = 0.0006) and in OS (9.1 months vs. 27.2 months, p = 0.01) were observed when the lowest quartile of CRBN expression was compared to the top three quartiles. Values used for the cut offs are as follows: (25%: 0.889687; 50%: 1.026542, 75%: 1.211133). While this study demonstrates a positive correlation between CRBN expression and response to pomalidomide and dexamethasone, an important caveat with respect to PFS and OS is that CRBN mRNA level is primarily a reflection of the number of copies of CRBN. Since focal deletions are uncommon, this mostly reflects chromosome 3p copy number. Since trisomy 3 is common in hyperdiploid, good prognosis disease, CRBN not only is required for IMiD function but higher levels also serve as a surrogate for low risk disease. Optimal gene expression cut offs for survival were determined using the Contal and O'Quigley method. The optimal cut point for PFS was 1.18443 and for OS was 1.17816. Conclusion: In summary, this study demonstrates, for the first time, a clinical correlation between CRBN expression and response to IMiD and dexamethasone therapy. CRBN expression is also predictive of survival outcomes and when quantified prior to therapy could potentially be utilized as a predictive biomarker for response to an IMiD based treatment. Disclosures: Stewart: Celgene: Consultancy, Honoraria.

Abstract 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.

Abstract FAM46C is one of the most recurrently mutated genes in multiple myeloma; however its role in disease pathogenesis has not been determined. Here we demonstrate that wild-type (WT) FAM46C overexpression induces substantial cytotoxicity in multiple myeloma cells. In contrast, FAM46C mutations found in multiple myeloma patients abrogate this cytotoxicity, indicating a survival advantage conferred by the FAM46C mutant phenotype. WT FAM46C overexpression downregulated IRF4, CEBPB, and MYC and upregulated immunoglobulin (Ig) light chain and HSPA5/BIP. Furthermore, pathway analysis suggests that enforced FAM46C expression activated the unfolded protein response pathway and induced mitochondrial dysfunction. CRISPR-mediated depletion of endogenous FAM46C enhanced multiple myeloma cell growth, decreased Ig light chain and HSPA5/BIP expression, activated ERK and antiapoptotic signaling, and conferred relative resistance to dexamethasone and lenalidomide treatments. Genes altered in FAM46C-depleted cells were enriched for signaling pathways regulating estrogen, glucocorticoid, B-cell receptor signaling, and ATM signaling. Together these results implicate FAM46C in myeloma cell growth and survival and identify FAM46C mutation as a contributor to myeloma pathogenesis and disease progression via perturbation in plasma cell differentiation and endoplasmic reticulum homeostasis. Cancer Res; 77(16); 4317–27. ©2017 AACR.

Key Points IMiDs inhibit TrxR-mediated intracellular decomposition of H2O2 and caused oxidative stress in MM cells. MM cells with lower antioxidative capacity were more vulnerable to lenalidomide-induced H2O2 accumulation and its associated cytotoxicity.