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.

Prostate cancer is a highly heterogeneous disease and mortality is mainly due to metastases but the initial steps of metastasis have not been well characterized. We have performed integrative whole exome sequencing and transcriptome analysis of primary prostate tumor foci and corresponding lymph node metastases (LNM) from 43 patients enrolled in clinical trial. We present evidence that, while there are some cases of clonally independent primary tumor foci, 87% of primary tumor foci and metastases are descended from a common ancestor. We demonstrate that genes related to oxidative phosphorylation are upregulated in LNM and in African-American patients relative to White patients. We further show that mutations in TP53, FLT4, EYA1, NCOR2, CSMD3, and PCDH15 are enriched in prostate cancer metastases. These findings were validated in a meta-analysis of 3929 primary tumors and 2721 metastases and reveal a pattern of molecular alterations underlying the pathology of metastatic prostate cancer. We show that LNM contain multiple subclones that are already present in primary tumor foci. We observed enrichment of mutations in several genes including understudied genes such as EYA1, CSMD3, FLT4, NCOR2, and PCDH15 and found that mutations in EYA1 and CSMD3 are associated with a poor outcome in prostate cancer.

Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.

Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely underexplored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and three-dimensional (3D) collective invasion. We further identified JAG1 signaling as a previously unknown upstream activator of MYO10 expression in leader cells. Using live-cell imaging, we found that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 up-regulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning.

Multiple myeloma (MM) development is dependent upon critical interactions with the bone marrow (BM) niche.1 The contribution of catecholamines and adrenergic signaling from the highly innervated BM niche2 to MM development is under-explored. MM patients demonstrate an elevated conserved transcriptional response to adversity (CTRA), indicative of stress that correlates with poor survival.3 A retrospective study evaluating the effects of the non-selective b adrenergic receptor (AR) blocker propranolol in immunomodulatory drug-treated MM found propranolol to improve progression-free survival (PFS) and overall survival (OS).4 MM patients exhibit reduced megakaryocyte–erythrocyte progenitors (MEP) and increased monocytic myeloid-derived suppressor cells (MDSC) (CD14+HLADRlow) in the BM, suggestive of increased myeloid bias.5 Introduction of MM precursor monoclonal gammopathy of undetermined significance (MGUS) cells into humanized IL-6 MIS(KI)TRG6 mice promotes progression to MM, suggesting the sufficiency of extrinsic BM niche elements in fostering MM development.6 Consistent with this, administration of propranolol in MM patients undergoing hematopoietic stem cell transplant (HSCT) demonstrates a significant reduction of not only the CTRA response, but also marked reductions in myeloid lineage bias.3 How targeting adrenergic signaling regulates hematopoietic stem and progenitor cell (HSPC) commitment in MM remains poorly understood. Our study provides mechanistic rationale for the application of propranolol to resolve both microenvironmental and MM-specific tumor promoting biology.

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ T cells. Murine tumor models revealed that tumor-specific CD8+ T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ T cell activation in response to cancer is different from that of canonical CD8+ T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

Despite advances in the treatment of multiple myeloma in the past decades, the disease remains incurable, and understanding signals and molecules that can control myeloma growth and survival are important for the development of novel therapeutic strategies. One such molecule, CD86, regulates multiple myeloma cell survival via its interaction with CD28 and signaling through its cytoplasmic tail. Although the CD86 cytoplasmic tail has been shown to be involved in drug resistance and can induce molecular changes in multiple myeloma cells, its function has been largely unexplored. Here, we show that CD86 cytoplasmic tail has a role in trafficking CD86 to the cell surface. This is due in part to a PDZbinding motif at its C-terminus which is important for proper trafficking from the Golgi apparatus. BioID analysis revealed 10 PDZ domain-containing proteins proximal to CD86 cytoplasmic tail in myeloma cells. Among them, we found the planar cell polarity proteins, SCRIB and DLG1, are important for proper CD86 surface expression and the growth and survival of myeloma cells. These findings indicate a mechanism by which myeloma cells confer cellular survival and drug resistance and indicate a possible motif to target for therapeutic gain.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response–dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress–induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

In multiple myeloma, the t(11;14) translocation enriches for patients likely to respond to the Bcl2 inhibitor venetoclax. In this group of patients, 40% respond to single-agent venetoclax while up to 60% respond to the combination of venetoclax and dexamethasone [1, 2]. We have previously demonstrated that ex vivo functional profiling of venetoclax sensitivity can more accurately identify these venetoclax-responsive patients [3]. Here we report updated data on a larger cohort of patients who underwent ex vivo testing and were subsequently treated with venetoclax. We demonstrate that this 24-hour functional assay can rapidly predict patient responses to venetoclax that translate into improved progression-free survival (PFS).

TET methylcytosine dioxygenases are essential for the stability and function of regulatory T cells (Treg cells), which maintain immune homeostasis and self-tolerance and express the lineage-determining transcription factor Foxp3. Here, we use whole-genome analyses to show that the transcriptional program and epigenetic features (DNA modification, chromatin accessibility) of Treg cells are attenuated in the absence of Tet2 and Tet3. Conversely, the addition of the TET activator vitamin C during TGFβ-induced iTreg cell differentiation in vitro potentiates the expression of Treg signature genes and alters the epigenetic landscape to better resemble that of Treg cells generated in vivo. Vitamin C enhances IL-2 responsiveness in iTreg cells by increasing IL2Rα expression, STAT5 phosphorylation, and STAT5 binding, mimicking the IL-2/STAT5 dependence of Treg cells generated in vivo. In summary, TET proteins play essential roles in maintaining Treg molecular features and promoting their dependence on IL-2. TET activity during endogenous Treg development and potentiation of TET activity by vitamin C during iTreg differentiation are necessary to maintain the transcriptional and epigenetic features of Treg cells.