ETV4-Dependent Transcriptional Plasticity Maintains MYC Expression and Results in IMiD Resistance in Multiple Myeloma.

Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Transcriptional Heterogeneity Overcomes Super-Enhancer Disrupting Drug Combinations in Multiple Myeloma.

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. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Tumor burden limits bispecific antibody efficacy through T cell exhaustion averted by concurrent cytotoxic therapy.

BCMA-CD3-targeting bispecific antibodies (BsAb) are a recently developed immunotherapy class which shows potent tumor killing activity in multiple myeloma (MM). Here, we investigated a murine BCMA-CD3-targeting BsAb in the immunocompetent Vk*MYC and its IMiD-sensitive derivative Vk*MYChCRBN models of MM. The BCMA-CD3 BsAb was safe and efficacious in a subset of mice, but failed in those with high-tumor burden, consistent with clinical reports of BsAb in leukemia. The combination of BCMA-CD3 BsAb with pomalidomide expanded lytic T cells and improved activity even in IMiD resistant high-tumor burden cases. Yet, survival was only marginally extended due to acute toxicity and T cell exhaustion, which impaired T cell persistence. In contrast, the combination with cyclophosphamide was safe and allowed for a tempered pro-inflammatory response associated with long-lasting complete remission. Concurrent cytotoxic therapy with BsAb actually improved T cell persistence and function, offering a promising approach to patients with a large tumor burden.

Monosomic loss of MIR15A/MIR16-1 is a driver of multiple myeloma proliferation and disease progression.

The most common genetic abnormality in multiple myeloma (MM) is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in MM predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3, but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of MM spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in MM initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1. Monoallelic deletion of Mir15a/Mir16-1 but not Rb1 was sufficient to accelerate the development of monoclonal gammopathy in wildtype mice, and the progression of MM in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with MM.

Mate pair sequencing outperforms fluorescence in situ hybridization in the genomic characterization of multiple myeloma.

Fluorescence in situ hybridization (FISH) is currently the gold-standard assay to detect recurrent genomic abnormalities of prognostic significance in multiple myeloma (MM). Since most translocations in MM involve a position effect with heterogeneous breakpoints, we hypothesize that FISH has the potential to miss translocations involving these regions. We evaluated 70 bone marrow samples from patients with plasma cell dyscrasia by FISH and whole-genome mate-pair sequencing (MPseq). Thirty cases (42.9%) displayed at least one instance of discordance between FISH and MPseq for each primary and secondary abnormality evaluated. Nine cases had abnormalities detected by FISH that went undetected by MPseq including 6 tetraploid clones and three cases with missed copy number abnormalities. In contrast, 19 cases had abnormalities detected by MPseq that went undetected by FISH. Seventeen were MYC rearrangements and two were 17p deletions. MPseq identified 36 MYC abnormalities and 17 (50.0% of MYC abnormal group with FISH results) displayed a false negative FISH result. MPseq identified 10 cases (14.3%) with IgL rearrangements, a recent marker of poor outcome, and 10% with abnormalities in genes associated with lenalidomide response or resistance. In summary, MPseq was superior in the characterization of rearrangement complexity and identification of secondary abnormalities demonstrating increased clinical value compared to FISH.

Identification of lenalidomide resistance pathways in myeloma and targeted resensitization using cereblon replacement, inhibition of STAT3 or targeting of IRF4.

To understand immunomodulatory drug (IMiD) resistance in multiple myeloma (MM), we created isogenic human multiple myeloma cell lines (HMCLs) sensitive and resistant to lenalidomide, respectively. Four HMCLs were demonstrated to be resistant to all IMiDs including lenalidomide, pomalidomide, and CC-220, but not to Bortezomib. In three HMLCs (MM.1.SLenRes, KMS11LenRes and OPM2LenRes), CRBN abnormalities were found, including chromosomal deletion, point mutation, and low CRBN expression. The remaining HMCL, XG1LenRes, showed no changes in CRBN but exhibited CD147 upregulation and impaired IRF4 downregulation after lenalidomide treatment. Depletion of CD147 in XG1LenRes and three additional HMCLs had no significant impact on MM viability and lenalidomide response. Further analysis of XG1LenRes demonstrated increased IL6 expression and constitutive STAT3 activation. Inhibition of STAT3 with a selective compound (PB-1-102) re-sensitized XG1LenRes to lenalidomide. Since XG1LenRes harbors a truncated IRF4 that is not downregulated by lenalidomide, we targeted IRF4/MYC axis with a selective inhibitor of the bromodomain of CBP/EP300 (SGC-CBP30), which restored lenalidomide response in XG1LenRes. This strategy also appeared to be more broadly applicable as SGC-CBP30 could re-sensitize two resistant HMCLs with low but detectable CRBN expression to lenalidomide, suggesting that targeting CBP/E300 is a promising approach to restore IMiD sensitivity in MM with detectable CRBN expression.

IAP antagonists induce anti-tumor immunity in multiple myeloma.

The cellular inhibitors of apoptosis (cIAP) 1 and 2 are amplified in about 3% of cancers and have been identified in multiple malignancies as being potential therapeutic targets as a result of their role in the evasion of apoptosis. Consequently, small-molecule IAP antagonists, such as LCL161, have entered clinical trials for their ability to induce tumor necrosis factor (TNF)-mediated apoptosis of cancer cells. However, cIAP1 and cIAP2 are recurrently homozygously deleted in multiple myeloma (MM), resulting in constitutive activation of the noncanonical nuclear factor (NF)-κB pathway. To our surprise, we observed robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and in patients with relapsed-refractory MM, where the addition of cyclophosphamide resulted in a median progression-free-survival of 10 months. This effect was not a result of direct induction of tumor cell death, but rather of upregulation of tumor-cell-autonomous type I interferon (IFN) signaling and a strong inflammatory response that resulted in the activation of macrophages and dendritic cells, leading to phagocytosis of tumor cells. Treatment of a MM mouse model with LCL161 established long-term anti-tumor protection and induced regression in a fraction of the mice. Notably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all of the treated mice.

A Novel Interaction between Pyk2 and MAP4K4 Is Integrated with Glioma Cell Migration.

Glioma cell migration correlates with Pyk2 activity, but the intrinsic mechanism that regulates the activity of Pyk2 is not fully understood. Previous studies have supported a role for the N-terminal FERM domain in the regulation of Pyk2 activity as mutations in the FERM domain inhibit Pyk2 phosphorylation. To search for novel protein-protein interactions mediated by the Pyk2 FERM domain, we utilized a yeast two-hybrid genetic selection to identify the mammalian Ste20 homolog MAP4K4 as a binding partner for the Pyk2 FERM domain. MAP4K4 coimmunoprecipitated with Pyk2 and was a substrate for Pyk2 but did not coimmunoprecipitate with the closely related focal adhesion kinase FAK. Knockdown of MAP4K4 expression inhibited glioma cell migration and effectively blocked Pyk2 stimulation of glioma cell. Increased expression of MAP4K4 stimulated glioma cell migration; however, this stimulation was blocked by knockdown of Pyk2 expression. These data support that the interaction of MAP4K4 and Pyk2 is integrated with glioma cell migration and suggest that inhibition of this interaction may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion.

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells.

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

The heat shock protein 70 cochaperone hip enhances functional maturation of glucocorticoid receptor.

Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes. The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established. Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo. Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR). Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded. Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein. Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone. In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells.