Lenalidomide Resistance Research Articles

Myelodysplastic syndromes del(5q): Pathogenesis and its therapeutic implications.

Myelodysplastic syndromes (MDS) encompass a heterogeneous set of acquired bone marrow neoplastic disorders characterized by ineffective hematopoiesis within one or more bone marrow lineages. Nearly half of MDS patients carry cytogenetic alterations, with del(5q) being the most prevalent. Since its first description, del(5q) was consistently correlated with a typical clinical phenotype marked by anemia, thrombocytosis, and a low risk of evolving into acute leukemia. Presently, the World Health Organization (WHO) classification of myeloid neoplasms recognizes a specific subtype of MDS known as "myelodysplastic neoplasm with low blast and isolated del(5q)" identified by the sole presence of 5q deletion or in combination with one other abnormality excluding -7/del(7q). Several studies have sought to unravel the biological processes triggered by del(5q) in the development of MDS, revealing the involvement of various genes localized in specific regions of chromosome 5 referred to as common deleted regions (CDR). This intricate biological landscape makes the MDS cells with del(5q) exceptionally sensitive to lenalidomide. Several studies have confirmed the efficacy of lenalidomide in this context. Regrettably, the response to lenalidomide is not conclusive, prompting ongoing research into biological mechanisms that drive patients toward leukemia and strategies to circumvent lenalidomide resistance and disease progression.

P-348 ADAR1-regulated cytoplasmic dsRNA-immune sensing pathway is a novel mechanism of lenalidomide resistance in multiple myeloma

P-348 ADAR1-regulated cytoplasmic dsRNA-immune sensing pathway is a novel mechanism of lenalidomide resistance in multiple myeloma

C-FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma.

The immunomodulatory drug lenalidomide, which is now widely used for the treatment of multiple myeloma (MM), exerts pharmacological action through the ubiquitin-dependent degradation of IKZF1 and subsequent down-regulation of interferon regulatory factor 4 (IRF4), a critical factor for the survival of MM cells. IKZF1 acts principally as a tumour suppressor via transcriptional repression of oncogenes in normal lymphoid lineages. In contrast, IKZF1 activates IRF4 and other oncogenes in MM cells, suggesting the involvement of unknown co-factors in switching the IKZF1 complex from a transcriptional repressor to an activator. The transactivating components of the IKZF1 complex might promote lenalidomide resistance by residing on regulatory regions of the IRF4 gene to maintain its transcription after IKZF1 degradation. To identify unknown components of the IKZF1 complex, we analyzed the genome-wide binding of IKZF1 in MM cells using chromatin immunoprecipitation-sequencing (ChIP-seq) and screened for the co-occupancy of IKZF1 with other DNA-binding factors on the myeloma genome using the ChIP-Atlas platform. We found that c-FOS, a member of the activator protein-1 (AP-1) family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The genome-wide screening revealed the co-occupancy of c-FOS with IKZF1 on the regulatory regions of IKZF1-target genes, including IRF4 and SLAMF7, in MM cells but not normal bone marrow progenitors, pre-B cells or mature T-lymphocytes. c-FOS and IKZF1 bound to the same consensus sequence as the IKZF1 complex through direct protein-protein interactions. The complex also includes c-JUN and IKZF3 but not IRF4. Treatment of MM cells with short-hairpin RNA against FOS or a selective AP-1 inhibitor significantly enhanced the anti-MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP-1 inhibitor mitigated the lenalidomide resistance of MM cells. C-FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co-factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

Histone Methyltransferase NSD2 Activates PKCα to Drive Metabolic Reprogramming and Lenalidomide Resistance in Multiple Myeloma.

Aberrant glycolysis driven by NSD2-mediated upregulation of PKCα can be therapeutically exploited using metabolic inhibitors with lactate as a biomarker to identify high-risk patients who exhibit poor response towards IMiD-based regimens.

Abstract 3427: ICP-490 is a highly potent and selective IKZF1/3 degrader with robust anti-tumor activities against multiple myeloma and non-Hodgkin’s lymphoma

Abstract Background: Cereblon (CRBN)-targeting immunomodulatory drugs (IMiDs), i.e., thalidomide and lenalidomide, play pivotal roles in treating multiple myeloma (MM) patients. This class of drugs induce rapid ubiquitination and degradation of two lymphoid transcription factors (IKZF1 and IKZF3) by recruiting them to CRBN-CRL4 E3 ubiquitin ligase, which result in pleiotropic anti-tumor effects including autonomous tumor cell death and immune activation. However, prolonged IMiD treatment eventually becomes resistant and leads to tumor relapse in MM patients, mostly due to down-regulation of CRBN. All approved IMiDs also exert adverse effects such as teratogenicity, attributed to CRBN-mediated degradation of neo-substrates SALL4, PLZF1 and ΔNp63/TAp63. Here we report the development of a novel selective IKZF1/3 degrader ICP-490, which can robustly inhibit MM and NHL tumor growth, and overcome lenalidomide resistance. Results: ICP-490 induces potent, deep, and selective degradation of IKZF1 and IKZF3 with sub-nanomolar IC50, but no significant degradation of IKZF2, IKZF4, GSPT1, SALL4 and PLZF1. Cell viability assays reveal robust in vitro efficacies of ICP-490 against various MM and NHL(DLBCL) cell lines with nanomolar IC50. It also exhibits potent anti-proliferative activity in lenalidomide-resistant cell lines. In contrast to its tumor killing effect, ICP-490 shows no cytotoxicity against normal human cells, such as HEK293 and PBMC cells. ICP-490 induces IKZF1/3 degradation in distinct human lymphocyte lineages, leads to immune cell activation with elevated IL-2 and IFN-γ production and down-regulation of TNF-α in PBMC. The immune modulation activity of ICP-490 has also been illustrated in a combinatory treatment with daratumumab, where low dose of ICP-490 leads to robust induction of IL-2 and granzyme B, and much improved efficacy of daratumumab in MM cell line NCI-H929. In vivo efficacy studies have further confirmed the effectiveness of ICP-490 against MM and DLBCL xenografts, including MM1.S, MM1.R, NCI-H929 multiple myeloma lines, and TMD-8, WSU-DLCL2 DLBCL lines. ICP-490 also eradicates lenalidomide-resistant NCI-H929 xenografts. ICP-490 has overall favorable pharmacokinetic parameters with high oral bioavailability. Nonclinical safety evaluations of ICP-490 exhibit acceptable drug tolerability in SD rats and cynomolgus monkeys. Conclusions: ICP-490 is a highly potent, orally bioavailable, next-generation CRBN-targeting IMiD with selective IKZF1/3 degradation. In various MM and NHL tumor models, it demonstrates superior tumor killing activities than currently approved IMiDs, including overcoming lenalidomide resistance. ICP-490 is now in phase I clinical trial. Citation Format: Ruixia Liang, Yucheng Pang, Yingrui Han, Haipeng Xu, Zuopeng Wang, Richard Liu, Charles Ying Wang, Jason Bin Zhang, Xiangyang Chen, Davy Xuesong Ouyang. ICP-490 is a highly potent and selective IKZF1/3 degrader with robust anti-tumor activities against multiple myeloma and non-Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3427.

Management and Outcomes of Anti-CD38 Refractory Myeloma Patients: The Impact of Retreatment and of Subsequent Therapies

The management of refractory myeloma patients (pts) to anti-CD38-based therapies is challenging despite the increasing number of treatment options. In addition, the use of anti-CD38 monoclonal antibodies (MoAbs) in earlier lines of treatment increases the number of refractory pts to these drugs. Management of such pts is based either in the use of drugs with new mechanisms of action (if available) or in the retreatment with same agents or drug classes used in prior lines. Herein, we describe the outcomes of pts who failed on anti-CD38-containing therapy. The analysis included 183 pts with RRMM who progressed during therapy with a daratumumab- or isatuximab-based regimens (index therapy) and who received further therapy. Pts were treated in a single center (Department of Clinical Therapeutics, University of Athens, Greece) and started anti-CD38 therapy from 1/1/2015 to 31/12/2021. The pts received index anti-CD38 therapy after a median of 2 prior lines of therapy (range 1-10). Their median age was 68 years (range 35-89); 38% had prior ASCT, 61% were PI-, 71% lenalidomide- and 29% pomalidomide-refractory. Anti-CD38 was given as monotherapy (+/- dexa) in 50% of pts, while its combination with IMiD was given in 25% of pts and with PI in 25% of them. On ITT, ORR was 55% (CR: 5%, VGPR: 25%, PR:25%). Median PFS on anti-CD38 therapy for the whole cohort was 6.4 months; it was 5.5 months for anti-CD38 +/- dexa vs 7.4 months for anti-CD38 triplets (p=0.055). Post anti-CD38 progression (median 3 prior lines) all pts were PI and lenalidomide exposed, 82% were PI refractory, 75% lenalidomide refractory and 46% pomalidomide refractory. Treatment post anti-CD38 failure included PI-based regimens in 40% of pts (23% carfilzomib-based), 30% pomalidomide containing, 23% anti-CD38 triplet combinations, 15% belantamab mafodotin, 5% selinexor (+/-PI); 50% received a triplet and 50% a doublet. On ITT, ORR in the post anti-CD38 line was 43% and the median PFS was 6.4 months; PFS was 5.5 months for pts that had progressed on anti-CD38+/-dexa, post-anti-CD38 and 7.4 months for those progressing on anti-CD38-combinations (p=0.195). Given the high rates of PI and lenalidomide resistance, only pomalidomide sensitivity was associated with better PFS post anti-CD38, which remained poor (median 7.5 vs 5.2 months, p=0.007). Median PFS in the post anti-CD38 line was 4 months for those who received anti-CD38 containing regimens, 6.4 months for PI-based regimens (for carfilzomib-containing 6.7 months), 4.5 months for pomalidomide-based, 6 months for triplets (vs 6.8 for doublets) while it was 9.1 months (95% CI 4.5-13.9) for belantamab mafodotin and 3.7 months in the small group of selinexor treated pts. A PFS ≥12 months during index anti-CD38 therapy was the most important prognostic factor for PFS post anti-CD38 failure (median PFS post anti-CD38 of 11.3 months vs 5.2 months, p=0.001). This prognostic effect was independent of the number of prior lines, type of treatment or pomalidomide resistance. Notably, the use of anti-CD38 combinations was associated with a PFS of 11.6 months in pts with a PFS ≥12 months during initial anti-CD38 therapy. The median OS post anti-CD38 progression was 17.6 months; 12.8 vs 22.5 months for pts who had progressed on antiCD38+/-dexa vs antiCD38 combinations respectively (p=0.327), The median OS post index therapy was 16.6 months for those who received anti-CD38 containing regimens, 22.9 months for PI-based regimens (for carfilzomib 22.7 months), 20.9 months for pomalidomide-based therapies, 21 months for triplets (vs 19.3 for doublets), 24.5 months for belantamab and 30 months for the small group of selinexor-treated pts. OS post anti-CD38 was 39 months for those with PFS ≥12 months on index antiCD38 therapy (vs 12 months, p<0.001) and this was the only important prognostic factor. In conclusion, outcomes after progression to anti-CD38 therapy are poor, despite recent therapeutic advances. Drugs with new mechanisms of action may improve PFS, but their use needs to be optimized, probably in the context of triplet combinations. Recycling prior therapies, even anti-CD38 drugs, may be beneficial especially for pts who had prolonged remission during the index anti-CD38 treatment and could be useful as a bridge to other options. In this subgroup of pts, maintaining an antiCD38 backbone could be a valid option but participation in clinical trials should be highly encouraged. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SORT1/LAMP2-mediated extracellular vesicle secretion and cell adhesion are linked to lenalidomide resistance in multiple myeloma

AbstractMultiple myeloma (MM) is a hematopoietic malignancy whose prognosis has improved with the development of new agents such as lenalidomide over the last decade. However, long-term exposure to drugs induces the acquisition of resistance by MM cells and leads to treatment failure and poor prognosis. Here, we show the molecular and cellular mechanisms of lenalidomide resistance in MM. In a comparison between lenalidomide-resistant cell lines and the parental cell lines, extracellular vesicle (EV) secretion and adherence abilities were significantly elevated in the resistant cells. Whole-transcriptome analysis revealed that the SORT1 and LAMP2 genes were key regulators of EV secretion. Silencing of these genes caused decreased EV secretion and loss of cell adhesion in the resistant cells, resulting in increased sensitivity to lenalidomide. Analysis of publicly available transcriptome data confirmed the relationship between genes related to EV secretion and cell adhesion and patient prognosis. Together, our findings reveal a novel mechanism of lenalidomide resistance in MM mediated by EV secretion and cell adhesion via SORT1 and LAMP2.

Loss of COP9 Signalosome Gene-Containing 2q Region Is Associated with Lenalidomide and Pomalidomide Resistance in Myeloma Patients

IntroductionIdentification of the causes of, and biomarkers for, drug resistance in myeloma is important for understanding treatment failures, and for future instigation of targeted therapeutics for myeloma. Using the largest set of whole genome sequencing (WGS) of advanced and drug resistant multiple myelomas to date, we reported that even heterozygous loss of the 3p region, which harbours immunomodulatory drug (IMiD) and CRBN E3 ligase modulator drug (CELMoD)-binding protein Cereblon (CRBN), undergoes strong therapeutic selection on lenalidomide (LEN) and/or pomalidomide (POM) treatment (Gooding et al 2021, PMC7893409). We hypothesized that copy loss of other genes required for IMiD activity may also have clinical relevance. Several groups have reported pharmacogenetic screens identifying genes essential for IMiD sensitivity in vitro, particularly genes required for the maintenance of the CUL4-DDB1-CRBN E3 Ubiquitin Ligase, such as members of the COP9 signalosome complex, function of which prevents CRBN protein degradation. However, loss of these genes has hitherto not been reported in myeloma.Methods and resultsWe identified candidate genes whose loss may favor IMiD drug resistance from published pharmacogenetic screens (n=5), and shortlisted genes consistently identified as essential for LEN or POM function in ≥2 screens (n=23). In our WGS dataset of 455 patients (cohorts: newly diagnosed (ND) n = 198, LEN-refractory n = 203; and LEN-then-POM-refractory n = 54), the incidence of mutation of shortlisted LEN/POM-essential genes in drug-refractory cohorts was rare (<5% combined), as found with CRBN. We next identified all those with overall incidence of >10% copy loss at the LEN-then-POM-refractory state, plus incidence of copy loss that increased from ND to LEN-then-POM-refractory states by ≥1.5-fold. This delivered 3 copy loss regions for further investigation: a) 3p, which we had already reported; b) 17p, loss of which is known to be strongly selected in myeloma as the site of TP53; and c) 2q, previously unidentified as relevant in myeloma, but whose minimal common region contained two members of the COP9 signalosome (COPS7B, COPS8). Proportion of loss of this region increased between ND (5.5%), LEN-refractory (9.8%) and LEN-then-POM-refractory states (16.6%), p=0.009. Those patients who had lost a copy of these genes also demonstrated a significant reduction in COPS7B/COPS8 gene expression (p<0.01 both genes).In a separate cohort of myeloma patients (n=24) with sequential sample WGS analysis before and after LEN and/or POM resistance acquisition, we traced acquisition of CNA-defined subclones. 5/24 (21%) patients had acquired either clonal or subclonal loss of the 2q region containing COPS7B and COPS8 at IMID resistance, which had been either absent or below limit of detection pre-IMiD exposure. No other CNA newly-emerged in such a high proportion during IMiD treatment. Relative decrease in even one COP9 signalosome gene has been shown to cause CRBN protein level to fall, and reduce LEN efficacy (Sievers et al 2018, PMC6148446). We are now analysing CRBN protein levels in sequential biopsies from these cases.ConclusionCopy number aberrations have not previously been shown to drive a therapy-specific clonal advantage in myeloma in the clinic. We have now identified a second novel CNA, 2q loss, which increases in incidence through LEN- and POM-refractory states to emerge as a marker of dominant clones in advanced, IMiD-resistant disease. Whether these CNAs will mark resistance to novel CELMoDs remains to be seen. The CRBN protein is key to the function of these drugs, and many novel proteolysis targeting chimeras (PROTACs) in development, but whether the kinetics of their CRBN binding are as sensitive to relative CRBN protein loss remains a key question. CNAs may be easily and cost-effectively detected in the clinic by targeted sequencing approaches, and may prove valuable in future therapeutic decision making. DisclosuresGooding: Bristol Myers Squibb: Research Funding. Ansari-Pour: Bristol Myers Squibb: Consultancy. Karagoz: h.: Research Funding. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Yong: Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding. Vyas: Astellas: Consultancy, Honoraria; Takeda: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Efficacy and Safety of Ruxolitinib and Steroids for Treating Patients with Relapsed or Refractory Multiple Myeloma

IntroductionRuxolitinib (RUX) is an FDA-approved oral, selective inhibitor of Janus kinase (JAK) 1/2 for the treatment of patients with intermediate or high-risk myelofibrosis and polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. Preclinical studies from our laboratory have demonstrated that RUX in combination with dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Specifically, RUX blocks expression of Mucin 1 whose function is linked to lenalidomide (LEN) resistance, downregulates PD-L1 and PD-L2 expression and reduces tumor stimulatory M2 macrophage polarization in multiple myeloma (MM) bone marrow. Recently published results from a Phase 1 trial for 28 heavily previously treated MM patients administered RUX, LEN and methylprednisolone (MP) demonstrated that the therapy was well tolerated and RUX overcame refractoriness to LEN and steroids (Berenson et al., Clin Cancer Res. 2020). The clinical benefit rate (CBR) and overall response rate (ORR) were 46% and 28%, respectively, and all 12 responding patients were refractory to LEN. We evaluated whether the combination of RUX and steroids without LEN was active for treating similar patients through treatment of a cohort within the current trial using this two-drug combination (NCT03110822).MethodsMM patients must have failed > 3 prior regimens and have been exposed to a proteasome inhibitor and LEN. Patients were treated orally (PO) with 15 mg RUX twice daily on days 1-28 of a 28-day cycle and 40 mg MP every other day. The treatment of this two-drug combination was continued until disease progression (PD). Once PD was confirmed, LEN at 10 mg PO daily on days 1-21 of a 28-day cycle was added to the regimen. Primary endpoints were safety, ORR and CBR.ResultsAs of July 15, 2021, 27 patients were enrolled. The median age was 64 years (range, 46-85), and 17 (63%) were male. Patients received a median of 4 (range, 3-11) prior treatments including LEN and steroid-containing regimens to which they were all refractory. Twenty-six patients have completed at least 1 full cycle of therapy; and, thus, were evaluable for efficacy.The ORR and CBR were 35% (n=9) and 39% (n=10), respectively. Notably, all 10 responding patients were refractory to LEN (i.e., progressed while on or within 8 weeks of last dosage). The remaining patients showed stable disease (n=12) or PD (n=4). The median follow-up was 13 months. The median progression-free survival was 4 months (range, 1-21). The median duration of response was 11 months (range, 1-20). Of 13 patients who progressed on the two-drug combination and had LEN added to their regimen, 6 patients responded (3 MR and 3 PR).Nine patients experienced SAEs including sepsis (12%), sepsis with neutropenic fever (4%), thrombocytopenia (4%), hyperglycemia (4%), neutropenia (4%), anemia (4%), acute heart failure (4%), rotator cuff tear (4%), osteomyelitis (4%), aspiration pneumonia (4%), pneumonia and pneumothorax (4%), and deep venous thrombosis (4%). Two patients died (one each from pneumonia and PD).ConclusionsThis ongoing Phase 1 trial is the first study demonstrating clinical activity of the two-drug combination of the JAK inhibitor RUX with steroids for MM patients. The treatment was well tolerated and shows promising efficacy for treating heavily previously treated MM patients. Because of this, additional studies are being conducted with higher doses of RUX in combination with MP for this patient population. DisclosuresVescio: Janssen: Speakers Bureau; Karyopharm: Speakers Bureau; GlaxoSnithKlein: Speakers Bureau; Amgen: Speakers Bureau. OffLabel Disclosure:Ruxolitinib is being investigated off-label to determine if it shows anti-myeloma activity in combination with steroids for relapsed/refractory myeloma patients.

Trial in Progress: A Perspective Study of Pan-Oral Triplet Regimen Pomalidomide, Ixazomib and Dexamethasone in Relapsed or Refractory Myeloma Patients

Background：During the past decade, bortezomib, lenalidomide and dexamethasone (VRd) followed by lenalidomide maintenance has become one of the most effective first-line regimens in multiple myeloma (MM) and been recommended by many guidelines. However, when disease relapses after VRd induction and lenalidomide maintenance, further generation of proteasome inhibitors or immunomodulator drugs, or agents with different mechanism should be considered. Pomalidomide, a new-generation oral immunomodulator drug, can overcome lenalidomide resistance and has better efficacy and safety in MM patients. Pomalidomide has a synergistic effect with proteasome inhibitor and dexamethasone, which has been proved more effective than pomalidomide and dexamethasone dual-drug regimen in relapsed or refractory MM (RRMM) patients (Richardson, et al. Lancet Oncol 2019). The overall response rate (ORR) of lenalidomide retreatment is only 54% (Sumit, et al. Blood 2011). A convenient and safe pan-oral regimen, ixazomib, pomalidomide and dexamethasone (IxaPd) is expected to improve the efficacy and survival in RRMM patients. The purpose of this study is to evaluate the efficacy and safety of IxaPd regimen in RRMM patients who have received lenalidomide in China.Study Design and Methods:This multi-center open-label, single-arm study (NCT04989140) will enroll patients with measurable M protein who have received 1 to 3 prior lines of therapy (including lenalidomide) with documented disease progression during or after their most recent treatment. Patients aged ≥18 years with Eastern Cooperative Oncology Group Performance Status 0-2, expected overall survival (OS) ≥3 months, and who provide informed consent will be eligible. Patients with prior exposure to pomalidomide, ixazomib and those intolerant/refractory to bortezomib will be excluded.Eligible 60 patients will be enrolled in this IxaPD regimen. Clinical and laboratory parameters will be recorded at baseline. Cytogenetic abnormalities are tested via fluorescence in situ hybridization (FISH) in CD138 sorted myeloma cells. Ixazomib 4 mg on days 1, 8 and 15 every 28 days, pomalidomide 4mg qd day 1-21 every 28-day, dexamethasone 40 mg (20 mg for patients >75 years of age) every week will be administrated. Treatment will continue until disease progression or unacceptable toxicity. Drug doses will be adjusted or withdrawn according to the degree of toxicities. The primary endpoint is progression free survival (PFS), the secondary endpoints include OS, time to next treatment (TTNT), ORR, safety. Patients are stratified into subpopulation on the basis of cytogenetic abnormalities, prior treatment lines and exposure drugs, ect. PFS, OS and TTNT were analyzed by Kaplan-Meier survival plot. Response will be evaluated according to International Myeloma Working Group (IMWG) 2016 criteria, and done at baseline and each cycle from 1-5, then every other cycle till cycle 13, and every three cycles till cycle 28 if the subjects do not relapse. Patients who reach complete response (CR) need to perform a minimal residual disease (MRD) test via next‐generation flow (NGF) .Conclusion:This is the first prospective clinical study to examine the efficacy and safety of pan-oral IxaPd regimen for RRMM in Chinese patients.DisclosuresResearch sponsor: CHIATAI TIANQING PHARMACEUTICAL GROUP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

OAB-032: Targeting GCK in RAS-mutant multiple myeloma offer a promising therapeutic approach

<h3>Background</h3> Next generation sequencing revealed frequent mutations of NRAS, KRAS or BRAF in up to 50% of newly diagnosed multiple myeloma (MM) patients. <h3>Methods</h3> Specific K- or N-RAS knockdowns led to strong decrease in MM cell viability if harbored the respective oncogenic isoform, but minimal effects were observed if only the wild-type isoform was present, presenting RAS signaling as the key driving oncogenes in the mutation bearing patients. Unfortunately, no inhibitor for RAS mutation is available for clinical use in MM. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK) is an upstream activator in the MAPK pathway. <h3>Results</h3> Our data showed that GCK (MAP4K2) knockdown in MM cells induced MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. Moreover, GCK silencing only led to significantly decreased GCK mRNA, however, did not affect IKZF1 expressions at mRNA level. We next tested the effects of GCK inhibitor TL4-12 on MM. Given the fact that RASMut MM cells have higher GCK expression level and are more sensitive to GCK knockdown, we compared mutated RAS MM cells with wild-type RAS MM cells. TL4-12 significantly inhibited the growth of mutated RAS MM cells, with IC50 5-10 fold lower compared to wild-type RAS MM cell lines. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Our data showed that GCK knockdown also downregulates IKZF1 protein level, indicating that IKZF1 is under the regulation of GCK. Concomitant treatment with proteasome inhibitor PS-341 blocked TL4-12 induced IKZF1 downregulation, suggesting that inhibition of GCK induces IKZF1 protein degradation. However, since IMiDs-resistant RPMI-8226 and JJN3 MM cells are sensitive to GCK inhibition, we hypothesized that the IKZF1 degradation mechanism induced by GCK inhibition is different from IMiDs and independent of CRBN. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells and examined the response to IMiDs and GCK inhibitor. CRBN silencing in H929 cells resulted in lenalidomide resistance. In contrast, CRBN silencing failed to rescue H929 MM from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. <h3>Conclusions</h3> Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibition downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance.

FOXM1 Is Associated with Development of Therapeutic Resistance in Multiple Myeloma

▪Despite advances in treatment resulting from development of novel therapies, multiple myeloma (MM) remains an incurable disease because of the emergence of therapeutic resistance. To explore mechanisms of acquired resistance to the IMiD® immunomodulatory agent lenalidomide in patients with MM, we performed RNA-seq analysis on 12 paired newly diagnosed MM (NDMM) patient samples of sorted CD138+ cells obtained prior to lenalidomide treatment initiation and after development of resistance. Gene set enrichment analysis using gene ontology biological process categories indicated that a dominant process associated with proliferation was upregulated at relapse. Transcription factor target enrichment analysis revealed that among differentially expressed genes, targets of forkhead box protein M1 (FOXM1), a transcription factor that promotes cell proliferation, were significantly (≈ 50% of FOXM1 target genes, false discovery rate [FDR] < 0.01) upregulated at relapse. FOXM1 expression also was significantly (FDR < 0.01) upregulated by 2.8-fold at relapse. Subsequently, we explored the association of FOXM1 gene expression with response to the IMiD immunomodulatory drug pomalidomide in MM patients relapsed/refractory (RRMM) to prior lenalidomide and proteasome inhibitor-based therapy. This analysis showed that patients with high baseline FOXM1 expression levels had lower response rates and shorter progression-free (median 2.9 vs 7.0 months; nominal logrank P < 2.24×10-6)and overall survival (median 10.4 vs 19.3 months; nominal logrank P < 8.43×10-7) when compared with patients with low baseline FOXM1 expression. Additional in vitro experiments indicated that knockdown of FOXM1 gene expression by short hairpin RNA increased sensitivity of a cell line intrinsically resistant to lenalidomide and pomalidomide, demonstrating an association between lower FOXM1 expression and IMiD agent sensitivity. Although FOXM1 expression has been linked to poor prognosis in NDMM, this work suggests high FOXM1 expression could also be associated with development of resistance to lenalidomide and cross resistance to pomalidomide in RRMM. DisclosuresAmatangelo:Celgene Corporation: Employment. Neri:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Towfic:Celgene Corporation: Employment, Equity Ownership; Immuneering Corporation: Equity Ownership. Bjorklund:Celgene Corporation: Employment. Kang:Celgene Corporation: Employment. Sternberg:Celgene Corporation: Employment. Bahlis:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Trotter:Celgene Institute for Translational Research Europe: Employment; Celgene Corporation: Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

ADAR1-MediatedGLI1 Editing Promotes Malignant Self-Renewal in Multiple Myeloma

IntroductionRepresenting ten percent of hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to therapy-resistant plasma cell leukemia (PCL). Despite many novel therapies, relapse rates remain high as a result of malignant regeneration (self-renewal) of MM cells in inflammatory microenvironments. In addition to recurrent DNA mutations and epigenetic deregulation, inflammatory cytokine-responsive adenosine deaminase associated with RNA (ADAR1) mediated adenosine to inosine (A-to-I) RNA editing has emerged as a key driver of cancer relapse and progression. In MM, copy number amplification of chromosome 1q21, which contains both ADAR1 and interleukin-6 receptor (IL-6R) gene loci, portends a poor prognosis. Thus, we hypothesized that ADAR1 copy number amplification combined with inflammatory cytokine activation of ADAR1 stimulate malignant regeneration of MM and therapeutic resistance.Methods and ResultsAnalysis of MMRF CoMMpass RNA sequencing (RNA-seq) data revealed that high ADAR1 expression (n=162 patients) correlated with significantly reduced progression-free and overall survival compared with a low ADAR1 subset (n=159 patients). In contrast to lentiviral ADAR1 shRNA knockdown and overexpression of an editase defective ADAR1 mutant (ADAR1E912A), lentiviral wild-type ADAR1 overexpression enhanced editing of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist. Editing of GLI1 transcripts enhanced GLI transcriptional activity in luciferase reporter assays, and promoted lenalidomide resistance in vitro . Finally, lentiviral shRNA ADAR1 knockdown reduced regeneration of high-risk MM in humanized serial transplantation mouse models indicative of reduced malignant self-renewal capacity. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and if selectively inhibited may prevent progression and relapse.ConclusionsDeregulated RNA editing, driven by aberrant ADAR1 activation, represents a unique source of transcriptomic and proteomic diversity, resulting in self-renewal of MM cells in inflammatory microenvironments. In summary, both genetic (1q21 amplification) and microenvironmental factors (inflammatory cytokines, IMiDs) combine to drive GLI1-dependent malignant regeneration in MM. Thus, ADAR1 represents both a vital prognostic biomarker and therapeutic target in MM. DisclosuresStewart:Amgen: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

Whole Exome Sequencing Reveals High Genetic Diversity in Relapsed Multiple Myeloma

Background: Treatments like lenalidomide and proteasome inhibitors alone or in combination with chemotherapy have dramatically improved the outcome in multiple myeloma in the past years. Although the majority of patients achieves a remission, almost all patients eventually relapse. Previous studies have found recurrent mutations in TP53 and FAM46C (Weinhold et al., Blood 2016) in relapsed multiple myeloma as well as CRBN mutations (Kortüm et al., Blood 2016) specifically in lenalidomide-treated patients.Methods: In order to identify additional genetic aberrations that are associated with therapy resistance, we analyzed CD138-selected multiple myeloma cells in bone marrow samples obtained pretreatment (n=12) and at different time points at progression during treatment (n=17) by fluorescence in situ hybridization (FISH) and whole exome sequencing (WES). Peripheral blood was used as a germline control. The median number of therapy lines between pretreatment and progression samples was 1 (range, 1 - 4). Progression samples were obtained after treatment with lenalidomide (n=16), bortezomib (n=14), or high-dose melphalan/autologous stem cell transplantation (n=9) alone or in combination or as sequential treatment. WES was performed by paired-end sequencing (read length 100 bp) on an Illumina HiSeq platform. The mean coverage was 117x. A variant allele frequency (VAF) of 5% was set as a cut-off for mutation calling.Results: In the pretreatment samples, the median number of cytogenetic aberrations was 2 (range, 1 - 4) and the median number of somatic variants was 39 (range, 24 - 63) per sample. There was no significant change in these numbers in posttreatment samples: median number of cytogenetic aberrations 2 (range, 0 - 4), median number of somatic variants 52 (range, 11 - 107).The most recurrent cytogenetic alterations at diagnosis were +1q21 (n=6), +9q34.2 (n=4), and del13q14 (n=3). At relapse, 13 of the 18 cytogenetic abnormalities detected at diagnosis were still present, 5 were lost and 5 were newly acquired. The most frequently lost aberration was +9q34.2 (n=2) and the most commonly acquired aberrations were +1q21 (n=2) and del13q14 (n=2). In pretreatment samples we found recurrent mutations in NRAS (n=4), KRAS (n=3), DIS3 (n=3), and IGLL5 (n=3). The median stability of mutations between pre- and posttreatment samples was 62% (range, 13 - 81%). NRAS mutations were lost in 2 progression samples and DIS3 in 1 progression sample while KRAS mutations remained stable. We found 430 relapse-specific mutations in the 17 posttreatment samples. Mutations in TP53 (n=4) were the most frequently newly acquired genetic events in progression samples, which is consistent with previous studies. Of the newly acquired gene mutations, only eight genes were affected in more than one progression sample, all other gene mutations were non-recurrent. Among genes involved in the mechanism of lenalidomide we found a DDB1 mutation in one progression sample after lenalidomide treatment. Another patient had an IKZF3 mutation at relapse. However, the mutation was located outside the lenalidomide degron sequence and the VAF was only 5%, suggesting that it was not responsible for lenalidomide resistance in the major clone. No relapse-specific mutations were found in other genes encoding proteins involved in lenalidomide or proteasome inhibitor activity like IKZF1, IRF4, CRBN, CUL4A, CUL4B or proteasome subunits. Pathway analyses revealed that mutations in genes involved in WNT/beta catenin and KRAS signaling were enriched in the progression samples.Conclusions: In conclusion, we found a high degree of genetic heterogeneity in relapsed multiple myeloma. There were no obvious mutations in genes involved in lenalidomide or proteasome activity that might drive drug resistance. TP53 mutations and +1q21 were the most recurrent gene aberrations identified at relapse, however, these events may cause a general robustness of multiple myeloma cells to multiple treatment modalities and might not be treatment specific. These results suggest that the genetic basis for drug resistance is heterogeneous and warrants genetic characterization of further patients by DNA and RNA sequencing as well as functional analyses of candidate genes and pathways. DisclosuresKronke:Celgene: Honoraria, Other: Travel Support; Takeda: Consultancy, Other: Travel Support.

Identifying Myeloma Patients at Risk of Lenalidomide or Pomalidomide Resistance at Relapse: A New Opportunity

Identifying Myeloma Patients at Risk of Lenalidomide or Pomalidomide Resistance at Relapse: A New Opportunity

Phase 1 open-label study of panobinostat, lenalidomide, bortezomib\u2009+\u2009dexamethasone in relapsed and relapsed/refractory multiple myeloma

Additional therapeutic options are needed for relapsed and refractory multiple myeloma (RRMM). We present data from a phase 1b, open-label, dose-escalation study (NCT01965353) of 20 patients with RRMM (median age: 63 years [range: 50–77]) and a median of four prior regimens (range: 2–14); 85% had refractory disease (lenalidomide [80%]; bortezomib [75%]; lenalidomide and bortezomib [50%]). Patients received a median of six cycles (range: 1–74) of panobinostat (10 or 15 mg), lenalidomide 15 mg, bortezomib 1 mg/m2, and dexamethasone 20 mg (pano-RVd). Median follow-up was ~14 months. Six dose-limiting toxicities were reported (mostly hematological); maximum tolerated dose of panobinostat (primary endpoint) was 10 mg. Most common adverse events (AEs) were diarrhea (60%) and peripheral neuropathy (60%); all grade 1/2. Grade 3/4 AEs occurred in 80% of patients and included decreased neutrophil (45%), platelet (25%) and white blood cell (25%) counts, anemia (25%) and hypophosphatemia (25%). No treatment-related discontinuations or mortality occurred. In evaluable patients (n = 18), overall response rate was 44%, and clinical benefit rate was 61%. Median duration of response was 9.2 months; progression-free survival was 7.4 months; overall survival was not reached. Pano-RVd proved generally well-tolerated and demonstrated potential to overcome lenalidomide and/or bortezomib resistance.

Serum Soluble Syndecan-1 (ssCD138) Can Contribute to the Discrimination of Lenalidomide Resistant Multiple Myeloma (MM) Patients

Background Lenalidomide resistance is one of the main emerging therapeutical concerns in MM. SsCD138 levels were shown of prognostic significance in MM at diagnosis while their contribution during disease course has not been evaluated. Aims To evaluate ssCD138 possible prognostic contribution in MM patients treated with Lenalidomide. Patients and methods : Sixty-nine patients treated with Lenalidomide were included in the study. All received dexamethasone (RD) but one maintenance. Medical records were reviewed and frozen sera samples were tested after patient's informed consent. SsCD138 levels were determined by ELISA (Diaclone Research) according to the manufacturer's instructions. Median age of patients was 67 years with 50% women. Immunoglobulin type was IgG in 67% patients, IgA in 22%, Light-Chain in 10% and IgD in 1%. ISS 1, 2 and 3 were 37%, 24% and 39% respectively. ssCD138 were measured at RD initiation, best response and relapse. Median value was used as cut off and any concentrations above median were defined as "High". Median PostLenOS was 27mo, median time to next treatment (TNT) was 12,5mo. Time from RD initiation to the second relapse after RD (LenPFS2) was 24,5mo. Median number of previous treatment lines was 3. Forty healthy individuals were also tested. Statistical analysis was performed using SPSS software v.25. Results: Median ssCD138 was 234 ng/ml (15-841) at RD initiation, 97 ng/ml(12-1500) at plateau and 247(51-395) at relapse while it was 52 ng/ml (21-1070) in Healthy Individuals. Patients achieving VgPR and better had more than 50% reduction in ssynd-1 levels from treatment initiation to plateau (r=0,344, p=0,05). Patients with High ssynd-1 levels at RD initiation presented shorter LenPFS 2 (p=0,011) and TNT (p=0,018). Factors measured at RD initiation impacting on LenPFS2 were ssCD138 levels (HR:2 .p=0,025), abnormal LDH (HR:4,47 ,p=0,0001), ISS (HR=1,54, p=0,008) and b2-microglobulin&amp;gt;5.5mg/L(HR: 2,23, p=0,009), Bone Marrow Infiltration (BMINF)&amp;gt;60% (HR : 2,39, p=0,048), albumin&amp;lt;3.5g/dl (HR: 2,72, p=0,12). In univariate analysis for TNT predictors, ssCD138 levels (HR:2,32, p=0,009), ISS (HR:1,46, p=0,001), b2-microglobulin&amp;gt;5.55mg/L (HR:1,76, p=0,008) and Free Light Chain Ratio(FLCR)&amp;gt;100 (HR:2,08, p=0,000), albumin&amp;lt;3.5 g/dl (HR: 1,75, p=0,031) BMINF&amp;gt;60% (HR : 2,18, p=0,01), Hb&amp;lt;10 g/dl (ΗR:3,3, p=0,001) were significant. Variables of importance for PostLenOS were ISS (HR:1,4 , p=0,004) b2-microglobulin&amp;gt;5.5mg/L (HR:1.61 ,p= 0,031), FLCR&amp;gt;100 (HR:2,8 , p=0,0001) levels, albumin&amp;lt;3.5 g/dl (HR: 2,45, p=0,001), BMINF&amp;gt;60% (HR : 2,8, p=0,002). Based on the independent variables patients were assigned a score of 1 with both FLCR&amp;gt;100 and elevated ssCD138 levels and 0 with both these values normal. Patients with 1 point presented an estimated TNT of 4 Vs 14 months (p=0,001) and shorter LenPFS of 11 Vs 28 months (p=0,001). The estimated PostLenOS was 15 Vs 33 months in patients with 1 and 0 point respectively (p=0,005). Conclusion SsCD138 levels in MM patients treated with RD were significantly associated with LenPFS2 and TNT. A Prognostic score based on ssCD138 and FLCR&amp;gt;100 was shown effective for the prediction of PostLenOS, LenPFS2 and TNT. These results suggest that soluble syndecan-1 levels can discriminate patients at risk to the lenalidomide resistant early. Indeed validation in larger cohort is necessary. Disclosures Panayiotidis: ASH: Honoraria; Phizer: Honoraria; Roche: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Takeda: Honoraria; Novartis: Honoraria, Research Funding; Genesis: Honoraria, Research Funding; Janssen: Honoraria; Gilead: Honoraria. Kyrtsonis:Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Efficacy and Safety of Ruxolitinib and Steroids for Treating Patients with Relapsed or Refractory Multiple Myeloma (RRMM)

Introduction Ruxolitinib (RUX) is an FDA-approved oral, selective inhibitor of Janus kinase (JAK) 1/2 for the treatment of patients with intermediate or high-risk myelofibrosis and polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. Preclinical studies from our laboratory have demonstrated that RUX in combination with dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Specifically, RUX blocks expression of Mucin 1 whose function is linked to lenalidomide (LEN) resistance and down-regulates PD-L1 and PD-L2 expression and reduces tumor stimulatory M2 macrophage polarization in multiple myeloma (MM) bone marrow. Recently published results from a Phase 1 trial for 28 heavily previously treated MM patients administered RUX, LEN and methylprednisolone (MP) demonstrated that the therapy was well tolerated and RUX overcame refractoriness to LEN and steroids (Berenson et al., Clin Cancer Res. 2020). The clinical and overall response rates were 46% and 28%, respectively, and all 12 responding patients were refractory to LEN. To further evaluate whether RUX and steroids without LEN demonstrate clinical activity and its tolerability in the same patient population, an ongoing Phase I trial was expanded to also include a cohort of patients treated with this two-drug combination (NCT03110822). Methods MM patients must have failed &amp;gt; 3 prior regimens and have been exposed to a proteasome inhibitor and LEN. Patients were treated orally (PO) with 15 mg RUX twice daily on days 1-28 of a 28-day cycle and 40 mg MP every other day. The treatment of this two-drug combination was continued until disease progression (PD). Once PD was confirmed, LEN at 10 mg PO daily on days 1-21 of a 28-day cycle was added to the regimen. Primary endpoints were safety, clinical benefit rate (CBR) and overall response rate (ORR). Results As of July 25, 2020, 16 of the planned 29 patients have been enrolled, and 15 patients have completed at least 1 full cycle of therapy; and, thus, were evaluable for efficacy. The median age was 64 years (range, 46-77), and 9 (60%) were male. Patients received a median of 4 (range, 3-10) prior treatments including LEN and steroids to which they were all refractory. Of the 15 evaluable patients treated with RUX and steroids, the CBR and ORR were 53% (n=8) and 33% (n=5), respectively. With a median follow-up of 4.7 months, the median duration of response was 3.5 months (range, 1-11+). Five and 2 patients showed stable disease and progressive disease, respectively. Notably, all 8 responding patients were refractory to LEN (i.e. progressed while on or within 8 weeks of last dosage). Of 6 patients who had progressed on the two-drug combination and had LEN added to their regimen, 3 patients responded (2 MR and 1 PR). Four patients experienced SAEs including sepsis (7%), group B strep sepsis with neutropenic fever and nausea (7%), pneumonia and pneumothorax (7%), thrombocytopenia (7%), and hyperglycemia (7%). Two patients died (one each from pneumonia and progressive disease). Conclusions This ongoing phase 1 trial is the first study reporting clinical activity of the JAK inhibitor RUX with steroids for MM. These results demonstrate that RUX with only steroids is also well tolerated and shows promising efficacy for treating heavily previously treated MM patients. Because of this, additional studies are being conducted with higher doses of RUX in combination with MP for this patient population. Disclosures Berenson: Takeda: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Incyte: Honoraria, Patents &amp; Royalties: OncoTracker, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Swift:Jassen: Consultancy, Honoraria, Speakers Bureau; bristol myers squibb: Consultancy, Honoraria, Speakers Bureau; Amgent: Consultancy, Honoraria, Speakers Bureau. Boccia:Incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Rigel: Consultancy, Honoraria, Research Funding, Speakers Bureau. OffLabel Disclosure: Ruxolitinib and steroids only for treatment patients with Multiple Myeloma

Gck Inhibition Is a Novel Therapeutic Strategy for RAS Mutated Multiple Myeloma and Overcomes Resistance to IMiDs

Introduction: RAS oncogenes are the most frequently mutated gene family in human cancers. 50% of newly diagnosed multiple myeloma patients carry a RAS/MAPK pathway mutation, with a rising percentage in the relapsed situation1. Thus, targeting RAS mutations in multiple myeloma will increase therapeutic efficiency and potentially overcome drug-resistance. Unfortunately, RAS mutations have been considered "undruggable" due to a lack of traditional small molecule binding pockets on the proteins. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK), also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), is an upstream activator in the MAPK pathway. Indeed, we recently discovered the critical role of GCK in RAS mutated (RASmut) MM cell survival and growth. GCK knockdown in RASmut MM cells induced MM cell growth inhibition both in vitro and in vivo. However, the detailed mechanism is yet to be defined. Methods and Results: Our previous data showed that GCK knockdown induces MM cell growth inhibition, associated with the blockage of MKK4/7-JNK phosphorylation and the downregulation of critical transcriptional factors (TFs) including IKZF1/3, BCL-6, and c-MYC proteins. To confirm that GCK knockdown downregulates IKZF1/3 etc at protein level but not mRNA level, we conducted real-time PCR on GCK knockdown MM cells and compared the expression of GCK and TFs to the empty vector (EV) infected MM cells. Results showed that shRNA induced GCK silencing only led to the significantly decreased GCK mRNA, however, did not affect IKZF1 and c-MYC expressions at mRNA level. Consistent with the effects of GCK knockdown, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 proteins, inhibited MM cell proliferation and induced cell apoptosis. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Importantly, our data showed that IMiDs-resistant RPMI-8226 MM cells have high expression of GCK. GCK knockdown and inhibition induced IKZF1 downregulation, triggered growth inhibition and cell apoptosis in RPMI-8226 cells, suggesting that GCK regulates IKZF1 degradation via a CRBN-independent mechanism. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells by shRNA lentiviral infection and examined the response to IMiDs and GCK inhibitor. CRBN knockdown was confirmed by western blotting. CRBN silencing in H929 cells resulted in lenalidomide (LEN) resistance, evidenced by the WTS proliferation assay. In contrast, CRBN silencing failed to rescue N-Rasmut H929 MM cells from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. Conclusion: Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibitor downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance. GCK inhibitors may represent a novel therapy for the treatment of RASmut MM patients, especially those who are resistant to IMiDs as well as with refractory or relapsed MM. References Walker, B.A., et al. Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin Oncol33, 3911-3920 (2015). Disclosures Marcireau: Sanofi: Current Employment. Lentzsch:Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months; Janssen: Consultancy; Sorrento: Consultancy; Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celularity: Consultancy; Magenta: Current equity holder in private company; Sanofi: Research Funding.

ARID2 is a pomalidomide-dependent CRL4CRBN substrate in multiple myeloma cells.

The immunomodulatory drug (IMiD) thalidomide and its derivatives lenalidomide and pomalidomide are therapeutic agents used in the treatment of multiple myeloma. Although pomalidomide offers considerable clinical benefits to patients with lenalidomide-resistant multiple myeloma, the molecular mechanisms underlying its superior efficacy remain unclear. Here we show that ARID2, a component of the polybromo-associated BAF (PBAF) chromatin-remodeling complex, is a pomalidomide-induced neosubstrate of CRL4CRBN. BRD7, another subunit of PBAF, is critical for pomalidomide-induced ARID2 degradation. ARID2 is involved in transcriptional regulation of pomalidomide target genes including MYC. Pomalidomide is more effective than lenalidomide in degrading ARID2 and is capable of inhibiting MYC expression and proliferation in lenalidomide-resistant cell lines. Notably, ARID2 expression is associated with a poor prognosis and is higher in chemoresistant minimal residual disease (MRD) populations, and in patients with relapsed/refractory multiple myeloma. These findings suggest that ARID2 is a promising target for overcoming lenalidomide resistance in patients with multiple myeloma.