Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance. Significance: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM.

The BCL2 inhibitor venetoclax promotes apoptosis in blood cancer cells and is approved for treatment of chronic lymphocytic leukemia and acute myeloid leukemia. However, multiple myeloma cells are frequently more dependent on MCL-1 for survival, conferring resistance to venetoclax. Here we report that mevalonate pathway inhibition with statins can overcome resistance to venetoclax in multiple myeloma cell lines and primary cells. In addition, statins sensitize to apoptosis induced by MCL-1 inhibitor, S63845. In retrospective analysis of venetoclax clinical studies in multiple myeloma, background statin use was associated with a significantly enhanced rate of stringent complete response and absence of progressive disease. Statins sensitize multiple myeloma cells to venetoclax by upregulating two proapoptotic proteins: PUMA via a p53-independent mechanism and NOXA via the integrated stress response. These findings provide rationale for prospective testing of statins with venetoclax regimens in multiple myeloma. Significance: BH3 mimetics including venetoclax hold promise for treatment of multiple myeloma but rational combinations are needed to broaden efficacy. This study presents mechanistic and clinical data to support addition of pitavastatin to venetoclax regimens in myeloma. The results open a new avenue for repurposing statins in blood cancer.

Treatment of refractory and/or relapsed multiple myeloma has been a challenging problem for over 20 years. However, we have made significant progress addressing this disease with the use of bortezomib, the first in class proteasome inhibitor, and the immunomodulatory agents, thalidomide and lenalidomide. Carfilzomib, the second-generation proteasome inhibitor, has also been approved for treatment of relapsed/refractory multiple myeloma. Carfilzomib is a highly selective and potent inhibitor of proteasome chymotrypsin-like activity. Phase I and II clinical trials have reported an acceptable toxicity profile, with manageable thrombocytopenia and anemia being the most common side effects. Peripheral neuropathy, a frequent dose-limiting side effect of bortezomib, was rare. Further, carfilzomib demonstrated encouraging single-agent activity and appeared to be effective even in patients refractory to bortezomib. Based on these promising data, carfilzomib is moving forward into Phase III trials for relapsed multiple myeloma and is also being investigated as front-line combination therapy for patients with newly diagnosed myeloma.

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape.

Although prognosis for patients with multiple myeloma has improved over the past decade, research toward discovery of new therapeutic avenues is important and could lead to a cure for this plasma cell malignancy. Here we show that blocking the CD28-CD86 pathway via silencing of either CD28 or CD86 leads to myeloma cell death. Inhibiting this pathway leads to downregulation of integrins and IRF4, a known myeloma survival factor. Our data also indicate that CD86, the canonical ligand in this pathway, has prosurvival activity that is dependent on its cytosolic domain. These findings indicate that targeting of this pathway is a promising therapeutic avenue for myeloma, because it leads to modulation of different processes important in cell viability.

Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. PDB ID codes: Comp. 2: 5IEZ; Comp. 5: 5IF4.

Protein homeostasis is critical for maintaining eukaryotic cell function as well as responses to intrinsic and extrinsic stress. The proteasome is a major portion of the proteolytic machinery in mammalian cells and plays an important role in protein homeostasis. Multiple myeloma (MM) is a plasma cell malignancy with high production of immunoglobulins and is especially sensitive to treatments that impact protein catabolism. Therapeutic agents such as proteasome inhibitors have demonstrated significant benefit for myeloma patients in all treatment phases. Here, we demonstrate that the 11S proteasome activator PA28α is upregulated in MM cells and is key for myeloma cell growth and proliferation. PA28α also regulates MM cell sensitivity to proteasome inhibitors. Downregulation of PA28α inhibits both proteasomal load and activity, resulting in a change in protein homeostasis less dependent on the proteasome and leads to cell resistance to proteasome inhibitors. Thus, our findings suggest an important role of PA28α in MM biology, and also provides a new approach for targeting the ubiquitin-proteasome system and ultimately sensitivity to proteasome inhibitors.

In a phase I clinical trial of Venetoclax in patients with t(11;14) relapsed/refractory MM, 40% of the patients treated achieved an objective response(1). While this response rate with a single agent in MM is impressive, it demonstrates that t(11;14) is not an optimal biomarker for response to venetoclax. Functional profiling of BCL2 family members has been shown to predict responses to therapy in myeloma and other diseases(2). Therefore, we set out to determine how ex vivo sensitivity to venetoclax corresponds to clinical response and resistance.

A major driver of multiple myeloma (MM) is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employed an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in MM to inform precision medicine strategies. Unbiased phosphoproteomics initially revealed differential activation of kinases across MM cell lines and that sensitivity to mammalian target of rapamycin (mTOR) inhibition may be particularly dependent on mTOR kinase baseline activity. We further noted differential activity of immediate downstream effectors of Ras as a function of cell line genotype. We extended these observations to patient transcriptome data in the Multiple Myeloma Research Foundation CoMMpass study. A machine-learning–based classifier identified surprisingly divergent transcriptional outputs between NRAS- and KRAS-mutated tumors. Genetic dependency and gene expression analysis revealed mutated Ras as a selective vulnerability, but not other MAPK pathway genes. Transcriptional analysis further suggested that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs wild-type RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes, whereas RAS mutations overall carry no survival impact. We further developed an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. These results will lead to improved stratification of MM patients in precision medicine trials while also revealing unexplored modes of Ras biology in MM.

Gain of chromosome 1q (+1q) is commonly identified in multiple myeloma and has been associated with inferior outcomes. However, the prognostic implication of +1q has not been evaluated in the setting of standard triplet regimens. We retrospectively analyzed 201 consecutive patients with newly diagnosed myeloma who received induction with lenalidomide, bortezomib, and dexamethasone (RVD) and were tested for +1q at diagnosis by fluorescent in-situ hybridization. Patients with +1q (n = 94), compared to those without +1q (n = 107), had shorter median progression-free survival (PFS) (41.9 months vs 65.1 months, p = 0.002, HR = 1.90) and overall survival (median not reached (NR) for either arm, p = 0.003, HR 2.69). In subgroup analyses, patients with co-occurring +1q and t(4;14), t(14;16) or del(17p) or with 4 or more copies of 1q had significantly worse PFS (25.1 months and 34.6 months, p < 0.001 and p = 0.0063, respectively), whereas patients with three copies and no other high-risk cytogenetic abnormalities had no significant difference in PFS. These data suggest that when treated with RVD induction, patients with +1q should be considered at very high risk for early progression in multiple myeloma when ≥4 copies are detected or in the context of other high-risk cytogenetic abnormalities.