Jessica Schmidt

Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide

The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.

Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides

The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

Identification of cereblon-binding proteins and relationship with response and survival after IMiDs in multiple myeloma

Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). Using 2 different methodologies, we identified 244 CRBN binding proteins and established relevance to MM biology by changes in their abundance after exposure to lenalidomide. Proteins most reproducibly binding CRBN (>fourfold vs controls) included DDB1, CUL4A, IKZF1, KPNA2, LTF, PFKL, PRKAR2A, RANGAP1, and SHMT2. After lenalidomide treatment, the abundance of 46 CRBN binding proteins decreased. We focused attention on 2 of these-IKZF1 and IKZF3. IZKF expression is similar across all MM stages or subtypes; however, IKZF1 is substantially lower in 3 of 5 IMiD-resistant MM cell lines. The cell line (FR4) with the lowest IKZF1 levels also harbors a damaging mutation and a translocation that upregulates IRF4, an IKZF target. Clinical relevance of CRBN-binding proteins was demonstrated in 44 refractory MM patients treated with pomalidomide and dexamethasone therapy in whom low IKZF1 gene expression predicted lack of response (0/11 responses in the lowest expression quartile). CRBN, IKZF1, and KPNA2 levels also correlate with significant differences in overall survival. Our study identifies CRBN-binding proteins and demonstrates that in addition to CRBN, IKZF1, and KPNA2, expression can predict survival outcomes.

Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-κB with antimyeloma activity in vitro and in vivo

As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-kappaB activation via inhibition of IKK alpha or IKK beta, whereas proteosome inhibitors instead suppress NF-kappaB function by impairing degradation of ubiquitinated I kappaB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-kappaB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kappaB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC(50) < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib--providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kappaB inhibitors as therapeutics for human multiple myeloma and related malignancies.

RNAi screen of the druggable genome identifies modulators of proteasome inhibitor sensitivity in myeloma including CDK5

The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6

A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein-coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

Identification of Molecular Vulnerabilities in Human Multiple Myeloma Cells by RNA Interference Lethality Screening of the Druggable Genome

Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non-multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma.

The clinical significance of cereblon expression in multiple myeloma

Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). We demonstrate here that no patient with very low CRBN expression responded to IMiD plus dexamethasone therapy. In 53 refractory MM patients treated with pomalidomide and dexamethasone, CRBN levels predict for decreased response rates and significant differences in PFS (3.0 vs. 8.9 months, p<0.001) and OS (9.1 vs. 27.2 months, p=0.01) (lowest quartile vs. highest three quartiles). While higher CRBN levels can serve as a surrogate for low risk disease, our study demonstrates that low CRBN expression can predict resistance to IMiD monotherapy and is a predictive biomarker for survival outcomes.

Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

Extramedullary myeloma whole genome sequencing reveals novel mutations in Cereblon, proteasome subunit G2 and the glucocorticoid receptor in multi drug resistant disease.

Extramedullary disease (EMD) in Multiple Myeloma (MM) is characterized by the detection of monoclonal plasma cells outside the bone marrow niche, and is frequently associated with poor prognosis. n nHere we describe novel genomic events leading to drug refractory disease in a heavily pretreated 37-year-old IgG-kappa MM patient presenting with progressive, multi-drug refractory EMD. For the first time we report an acquired truncating mutation of Cereblon (CRBN) as well as point mutations in proteasome subunit G2 and the glucocorticoid receptor as an explanation for drug resistance. Initial myeloma treatment for the patient occurred over multiple years and included the immunomodulatory drugs (IMiDs) thalidomide and lenalidomide, the proteasome inhibitor bortezomib, cortiosteroids, radiation, one autologous and two allogeneic transplantations. She experienced extramedullary relapse, presenting as an extensive neck mass and smaller soft tissue nodules in the upper left triceps. The most recent therapy immediately prior to genomic sequencing was hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) incorporating alkylating agent cyclophosphamide with transient minor response. The patient was then enrolled in a pilot study utilizing next generation sequencing (NGS) to identify novel markers and potential therapeutic targets. Samples were acquired with patient consent in compliance with Mayo Clinic Institutional Review Board. For this study we completed array comparative genomic hybridization, whole exome, whole genome (insert = 1 kb) and RNA sequencing (RNASeq) of a biopsy taken from the neck mass to thoroughly interrogate the tumour genome of this patient. The presence of mutations of interest was evaluated by capillary sequencing in an expanded cohort of 25 CD138+ MM samples with low CRBN expression. n nThe neck mass pathology confirmed sheets of atypical plasma cells, kappa light chain restriction, CD138+, CD20− and CD45−. Array comparative genomic hybridization revealed multiple copy number abnormalities, most notably del1(p13.2–34.2), monosomy 13 and monosomy X. Therapy subsequent to biopsy for genome sequencing was with pomalidomide and dexamethasone without response. Unfortunately, the patient succumbed to her disease in less than the 12 weeks required at the time for sequencing and data analysis. n nSequencing revealed a highly disturbed genome (Figure 1) consisting of 4 somatic insertions/deletions, 38 intra-chromosomal rearrangements, and 35 translocations, including the high risk marker and initiating tumour event t(14;16). Furthermore, 271 nonsynonymous, somatic point mutations were detected in genes including KRAS, PIK3CA, ATM, and NFKB2 (Table I). Importantly, a Q99* truncating mutation as well as a R283K point mutation were observed in CRBN, that we recently demonstrated as essential for the anti-MM action of IMiDs (Zhu, et al 2011). To our knowledge this is the first documented mutation of Cereblon in a primary myeloma sample. Additional sequencing of CRBN in the expanded cohort of 25 patients revealed a synonymous mutation in only one sample. n n n nFigure 1 n nCircos plot depicting genome wide somatic variants, rearrangements and copy number changes derived from next generation sequencing. Numbers with circles around them indicate the following: 1) somatic single nucleotide variation (SNV), 2) location of SNV ... n n n n n nTable I n nSummary of clinically relevant single nucleotide variations n n n nWe also observed in the patient biopsy a potentially clinically relevant nonsynonymous point mutation in proteasome assembly chaperone 2, PSMG2 (E171K). PSMG2 is a proteasome assembly protein involved in mammalian 20S proteasome maturation (Hirano, et al 2005). Mutations in proteasome assembly components contribute to proteasome inhibitor resistance (Keats, et al 2007), possibly explaining this patient’s bortezomib-refractory disease. Capillary sequencing of PSMG2 in our expanded cohort revealed no mutations, although exonic deletion of PSMG2 has also been reported in myeloma (Walker, et al 2012). n nThe last nonsynonymous point mutation associated with drug resistance was identified in NR3C1 (G369A), a glucocorticoid receptor. Mutation of NR3C1 has been associated with resistance to steroid therapy (Bray and Cotton 2003), which this patient received and proved refractory. No NR3C1 mutations were identified in our expanded cohort and none have been previously reported in other myeloma genomes (Chapman, et al 2011, Walker, et al 2012). Mutations in NR3C1 have however been described in the glucocorticoid resistant MM.1R cell line (Moalli, et al 1992). Patients with low NR3C1 expression levels who received thalidomide demonstrated better progression-free survival and overall survival than those with low NR3C1 who did not receive thalidomide (Heuck, et al 2012). n nWhile these mutations suggest causality of drug-refractory disease, they do not identify pathways that can be exploited with targeted therapies. Additional mutations were observed in pathways for which targeted therapies are available. This patient had mutations in KRAS (G12C) and in ATM (T1985I), both of which affect the signalling of MEK downstream, thus making MEK a therapeutic target of interest in this patient. While there are no approved MEK inhibitors available for MM treatment, more than 100 trials are currently investigating MEK inhibitors, of which three of these trials are being conducted in MM patients (www.clinicaltrials.gov). n nThe patient tumour also contained canonical, activating mutations in PIK3CA (E542K). Interestingly, one study demonstrated that 64% of PIK3CA mutations occur in exon 9, where codon 542 is located. Moreover, 19% of patients with PIK3CA mutations also presented with KRAS mutations, of which 9% are G12C (Janku, et al 2012), found in our patient. The PI3K pathway is vitally important as it regulates downstream targets, such as AKT and MTOR, which are responsible for cell proliferation, growth, survival and metastasis (Bartholomeusz and Gonzalez-Angulo 2012). In addition, a number of clinical trials are currently investigating PIK3 inhibitors (www.clinicaltrials.gov). n nIn summary, this is the first description of CRBN mutations in a primary myeloma sample and furthermore of a “triple negative” MM patient possessing mutations probably contributing to resistance to all three major drug classes utilized in MM therapy. These mutations were not replicated in our validation cohort of 25 patients with low level CRBN expression and functional data have not yet been obtained, thus further investigation is necessary to better understand the mutation frequency and the functional significance of mutation in these genes. In summary, our approach utilizing comprehensive next generation sequencing not only identified mutations suggestive of the patient’s refractory disease, but also revealed unforeseen therapeutic options highlighting the importance of this technology in advancing individualized medicine.