Citations

Abstract Background: The proteasome inhibitor Bortezomib (BTZ) is an efficient treatment option for both, newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite the effectiveness, most patients eventually acquire drug resistance for reasons not fully understood Materials and methods: To better understand BTZ resistance mechanism we used a CRISPR library (GeCKO V2) targeting 19052 human genes, trying to identify genes responsible for BTZ resistance. CRISPR sgRNAs targeting the ERN1-XBP1 pathway were used as positive controls. We first infected RPMI 8226 myeloma cell line expressing Cas9 with the CRISPR library packaged into lenti-vectors and selected for resistance to BTZ. Surviving cells were subjected to next generation sequencing. Based on the initial screen results we constructed a second CRISPR sgRNA library including 31 genes, each gene targeted with four sgRNAs. After the second round screening and subsequent sequencing, we selected the top 20 genes for individual validation. Results: Proteasome regulatory gene PSMC6 was identified as the only reproducible gene conferring BTZ resistance. Interestingly, the same gene was independently found by a second group using a CRISPR approach (Sheffer M et al. ASH Abstract 273, 2014). Resistance was reproducible using a PSMC6 knockout by three individual CRISPR sgRNAs targeting exonic regions and one pair of SgRNA targeting intron region flanking exon for the deletion of exon one. PSMC6 knockout was verified by PCR and Sanger sequencing. Sensitivity to BTZ was rescued by over expression of PSMC6 cDNA in RPMI 8226 cells harboring a deletion of PSMC6 exon 1. MM cells lacking PSMC6 also developed resistance against Carfilzomib. Resistance was reproduced on a second MM cell line, KMS11. We did not see any significant difference of toxicity in PSMC6 deleted cells for other chemicals tested (tunicamycin, staurosporine, dexamethasone and melphalan). We demonstrated that the sensitivity of chymotrypsin-like activity of proteasome against BTZ was significantly reduced in cells lacking PSMC6. Consequently, MM cells without the PSMC6 gene were relatively resistant to apoptosis induced by BTZ, which was verified by Western blot for caspase 8 degradation and luminescent assay for caspase 3/7 activities. Clinically we could not correlate the PSMC6 expression level with the outcome of BTZ treatment in BTZ naive patients using publically available gene expression data. We initially used CRISPR sgRNAs targeting ER stress pathway (ERN1 and XBP1) as a positive control. However we could not derive any resistant cells from the experiment. We also could not identify any sgRNAs targeting the ERN1-XBP1 pathway from our whole exome screen and next generation sequencing. Since this contradicts published data, we decided to knockout ERN1 and XBP1 genes individually. Clones of cells with successful knockout of ERN1 in three cell lines (RPMI 8226, KMS11 and JJN3) and XBP1 in two cell lines (RPMI 8226 and KMS11) were tested for response to BTZ and Carfilzomib , however we did not find any drug response difference between the knockout and parent cells. We also found that the ERN1-XBP1 knockout cells did not show difference in response to ERN1 specific inhibitors (4u8C and STF-038010) and ER stress inducers (tunicamycin and thapsigargin) compared to parental cells. Conclusions: Human multiple myeloma cells lacking the PSMC6 gene develop significant resistance to apoptosis induced by BTZ. We have however not found a correlation of PSMC6 expression levels with outcome to BTZ treatment in BTZ naïve patients. We are therefore currently investigating the PSMC6 mutation rate in relapsed MM patients after proteasome treatment. In contrast to previous reports showing that progenitor MM cells lacing XBP-1 or ERN-1 invoked BTZ resistance, we were not able to demonstrate a change in sensitivity after full CRISPR knock out of either ERN1 or XBP1. It has long been believed that ERN1-XBP1 pathway plays an important role for MM treatment, leading to the development of ERN1 specific inhibitors. However, we demonstrated that the toxicity of two ERN1-specific inhibitors appears independent of the ERN1-XBP1 pathway. We also demonstrated that the toxicity of two important ER stress inducers, tunicamycin and thapsigargin, is independent of the ERN1-XBP1 pathway. Disclosures Stewart: Celgene: Consultancy; Oncospire Inc.: Equity Ownership; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Abstract Background: It is now well established that the immune modulator (IMiD) drugs thalidomide (Thal), lenalidomide (Len) and pomalidomide (Pom) bind a specific pocket in the protein, cereblon (CRBN) that is required for MM cytotoxicity after IMiD treatment. Recently, we reported 46 CRBN binding proteins which were altered by Len, two of which were the transcription factors Ikaros (IKZF1) and Aiolis (IKZF3), important regulators of B and T cell development. Others had previously reported the same finding and went further by demonstrating that following binding of IMiD drugs, CRBN accumulates and accentuates the ubiquitination and subsequent proteasomal degradation of these two binding proteins. The resulting degradation and depletion of IKZF proteins has the simultaneous effect of downregulating IRF4 and MYC leading to MM cell death while simultaneously enhancing transcription of interleukin 2 and activating the immune compartment. Methods: To further explore this phenomenon and its relationship to drug sensitivity we first created two stable MM cell lines (H929IKZF1Luc, 8226IKZF1Luc) expressing an IKZF1-luciferase (luc) fusion gene and tested for sensitivity to Len alone and in combination with dexamethasone (Dex), bortezomib (Bor) or the histone deacetylase inhibitor SAHA. Next, we constructed an adenoviral vector expressing IKZF1-luc and used this to transfect 16 MM cell lines, confirmed expression and tested sensitivity to Len by MTT (Figure 1). Results: We first examined the stable cell lines and measured luc expression after exposure to all three IMiD drugs. With all IMiD drugs luc dramatically decreased in sensitive H929 cells. IKZF1 depletion was both time and dose dependent. Using this surrogate marker cell line assay, luc is most efficiently degraded by Pom which is twice as potent as Len, but a thousand times more potent than Thal (Concentration of drug required to reduce luc expression by 50%, Pom: 4.9nM, Len: 10.2 nM, and Thal: 4,795 nM). We then tested all 16 cell lines with Len 2uM for 5 hours and show a strong correlation of IKZF-luc depletion with sensitivity. Figure 1: Luc levels 5 hours after Len treatment at 2uM. We divided the cell lines into three groups: 1) Len resistant: 8226, KMS12PE, My5, SKMM2, JJN3, FR4, H112; 2) Len intermediate response: EJM, OPM2, U266, KMS18; 3) Len sensitive: H929, MM1.S, MM1.R, KMS11, XG1; Figure 1:. Luc levels 5 hours after Len treatment at 2uM. We divided the cell lines into three groups: 1) Len resistant: 8226, KMS12PE, My5, SKMM2, JJN3, FR4, H112; 2) Len intermediate response: EJM, OPM2, U266, KMS18; 3) Len sensitive: H929, MM1.S, MM1.R, KMS11, XG1; We next performed western blots on 9 MM cell lines and demonstrate that IKZF1 levels were lowest in 3 of 5 resistant MM cell lines and normal in all 4 sensitive lines studied. The cell line (FR4) with the lowest levels of IKZF1 harbors a damaging mutation of IZKF1 as well as a translocation which upregulates IRF4, an IKZF target. Of the remaining two resistant lines one has low levels of Cereblon. In one line the mechanism of IMiD resistance could not be established. Finally, we were curious about the dichotomy of the IMiD biology which seemingly has an absolute requirement for a functional proteasome for successful IMiD cytotoxicity which is at variance with clinical reports of synergy between the IMiDs and the proteasome inhibitors (PIs) Bor and carfilzomib (Car). To explore this, we treated the 8226IKZF1Luc cell line with Len alone or in combination with increasing concentrations of Bor (6 nM) or Car (40nM) and found that both totally blocked IKZF1 fusion protein degradation promoted by Len, beginning as soon as 1 hour at 100nM Bor or after 3 hours at 40nM treatment. Conversely, in low concentration of Bor (<4nM) no IKZF1 degradation blocking was observed in the Luc assay and Bor remained lethal to the MM cells. In that light with lower levels of Bor (<3nM) even varying the sequencing of MM cell exposure to the two drug classes did not block cytotoxicity. Other MM active drug Dex or SAHA, blocking the aggresome, had no significant inhibition for Len-induced IKZF1 fusion protein degradation. Conclusion: These results validate the central role for the CRBN-IKZF-IRF4 axis proteins in IMiD response and show a strong correlation of IZKF1 expression and the ability to degrade IKZF1 with sensitivity. Our data provide interesting and perhaps disconcerting biologic data suggesting that the timing and dosing of clinical use of IMiDs and PI treatment in combination should be carefully evaluated to prevent antagonism of effect but suggest in vitro that a therapeutic window exists at which proteasome inhibitors can be active without blocking IMiD activity. Disclosures Stewart: Sanofi: Consultancy; Array BioPharma: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding.

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 3968 To better understand the genetic events associated with extra-medullary multiple myeloma (MM) we completed whole exome, long-insert whole genome and RNA sequencing on a patient with t(14;16) and neck adenopathy, as well as whole genome sequencing on two additional patients with respectively: t(4;14) and plasma cell leukemia, and del 17 with chest wall mass. All patients had multi-drug resistant disease and del 13. The frequency of somatic non-synonymous single nucleotide variants (SNV) ranged from 119 to 498, including an average of 15 truncating SNV, as well an average of 75 structural variants (range 2–147). Importantly, in one patient a truncating mutation, Q99*, on one allele and minor clone point mutation, R283K, on the second allele was observed in CRBN, a gene whose expression is required for effective lenalidomide and pomalidomide anti-myeloma activity. In the same patient a non-synonymous mutation, E171K, was also observed in PSMG2, a proteasome assembly protein, and a non-synonymous mutation, G369A, in NR3C1, a glucocorticoid receptor. Subsequently we have sequenced CRBN in 27 patients and 7 cell lines and have found no further examples of mutation in CRBN but have previously reported a bi-alleleic focal deletion in the MM.1Sres cell line. Mutations in NR3C1 have not previously been described in other MM genomes. Other, potentially clinically relevant mutations in the three patients were observed in FGFR3, PRDM1, STAT5B, NRAS, KRAS, PIK3CA, NF1, ATM, NFKB2, CKS1B and ERBB4. Although the three patients did not share a unifying event, a number of mutated genes observed were shared with other published myeloma genomes including RB1, TP53, MLL3, JAG2, VAV3 and SETD2. We also looked at pathway activation across the three genomes with the most statistically mutated pathways including VEGF signaling and activation, cell adhesion and chemokines, cytoskeleton remodeling, integrins in carcinoma progression, TP53 signaling and NOTCH signaling. In conclusion, we have identified, for the first time, the presence of a mutation in CRBN in a myeloma patient as well as mutations in PSMG2 and NR3C1, likely associated with drug resistance. Disclosures: Schmidt: Karyopharm: Research Funding. Carpten:Life Technologies: Research Funding. 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.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F‐TKIs) can elicit meaningful objective clinical responses in patients carrying FF‐positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F‐TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2‐TACC3 (transforming acidic coiled‐coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2‐TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF‐driven ICC.

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.