Muhammad B. Ekram

JARID1B Is a Luminal Lineage-Driving Oncogene in Breast Cancer

Recurrent mutations in histone-modifying enzymes imply key roles in tumorigenesis, yet their functional relevance is largely unknown. Here, we show that JARID1B, encoding a histone H3 lysine 4 (H3K4) demethylase, is frequently amplified and overexpressed in luminal breast tumors and a somatic mutation in a basal-like breast cancer results in the gain of unique chromatin binding and luminal expression and splicing patterns. Downregulation of JARID1B in luminal cells induces basal genes expression and growth arrest, which is rescued by TGFβ pathway inhibitors. Integrated JARID1B chromatin binding, H3K4 methylation, and expression profiles suggest a key function for JARID1B in luminal cell-specific expression programs. High luminal JARID1B activity is associated with poor outcome in patients with hormone receptor-positive breast tumors.

HOXB7 Is an ERα Cofactor in the Activation of HER2 and Multiple ER Target Genes Leading to Endocrine Resistance

UNLABELLEDnWhy breast cancers become resistant to tamoxifen despite continued expression of the estrogen receptor-α (ERα) and what factors are responsible for high HER2 expression in these tumors remains an enigma. HOXB7 chromatin immunoprecipitation analysis followed by validation showed that HOXB7 physically interacts with ERα, and that the HOXB7-ERα complex enhances transcription of many ERα target genes, including HER2. Investigating strategies for controlling HOXB7, our studies revealed that MYC, stabilized via phosphorylation mediated by EGFR-HER2 signaling, inhibits transcription of miR-196a, a HOXB7 repressor. This leads to increased expression of HOXB7, ER target genes, and HER2. Repressing MYC using small-molecule inhibitors reverses these events and causes regression of breast cancer xenografts. The MYC-HOXB7-HER2 signaling pathway is eminently targetable in endocrine-resistant breast cancer.nnnSIGNIFICANCEnHOXB7 acts as an ERα cofactor regulating a myriad of ER target genes, including HER2, in tamoxifen-resistant breast cancer. HOXB7 expression is controlled by MYC via transcriptional regulation of the HOXB7 repressor miR-196a; consequently, antagonists of MYC cause reversal of selective ER modulator resistance both in vitro and in vivo.

Spatial proximity to fibroblasts impacts molecular features and therapeutic sensitivity of breast cancer cells influencing clinical outcomes

Using a three-dimensional coculture model, we identified significant subtype-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells in contact with cancer-associated fibroblasts (CAF). CAF-induced gene expression signatures predicted clinical outcome and immune-related differences in the microenvironment. We found that fibroblasts strongly protect carcinoma cells from lapatinib, attributable to its reduced accumulation in carcinoma cells and an elevated apoptotic threshold. Fibroblasts from normal breast tissues and stromal cultures of brain metastases of breast cancer had similar effects as CAFs. Using synthetic lethality approaches, we identified molecular pathways whose inhibition sensitizes HER2+ breast cancer cells to lapatinib both in vitro and in vivo, including JAK2/STAT3 and hyaluronic acid. Neoadjuvant lapatinib therapy in HER2+ breast tumors lead to a significant increase of phospho-STAT3+ cancer cells and a decrease in the spatial proximity of proliferating (Ki67+) cells to CAFs impacting therapeutic responses. Our studies identify CAF-induced physiologically and clinically relevant changes in cancer cells and offer novel approaches for overcoming microenvironment-mediated therapeutic resistance. Cancer Res; 76(22); 6495-506. ©2016 AACR.

Immune Escape in Breast Cancer During In Situ to Invasive Carcinoma Transition

To investigate immune escape during breast tumor progression, we analyzed the composition of leukocytes in normal breast tissues, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDC). We found significant tissue and tumor subtype-specific differences in multiple cell types including T cells and neutrophils. Gene expression profiling of CD45+CD3+ T cells demonstrated a decrease in CD8+ signatures in IDCs. Immunofluorescence analysis showed fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including in matched DCIS and recurrent IDC. T-cell receptor clonotype diversity was significantly higher in DCIS than in IDCs. Immune checkpoint protein TIGIT-expressing T cells were more frequent in DCIS, whereas high PD-L1 expression and amplification of CD274 (encoding PD-L1) was only detected in triple-negative IDCs. Coamplification of a 17q12 chemokine cluster with ERBB2 subdivided HER2+ breast tumors into immunologically and clinically distinct subtypes. Our results show coevolution of cancer cells and the immune microenvironment during tumor progression.Significance: The design of effective cancer immunotherapies requires the understanding of mechanisms underlying immune escape during tumor progression. Here we demonstrate a switch to a less active tumor immune environment during the in situ to invasive breast carcinoma transition, and identify immune regulators and genomic alterations that shape tumor evolution. Cancer Discov; 7(10); 1098-115. ©2017 AACR.See related commentary by Speiser and Verdeil, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

LSD1-Mediated Epigenetic Reprogramming Drives CENPE Expression and Prostate Cancer Progression

Androgen receptor (AR) signaling is a key driver of prostate cancer, and androgen-deprivation therapy (ADT) is a standard treatment for patients with advanced and metastatic disease. However, patients receiving ADT eventually develop incurable castration-resistant prostate cancer (CRPC). Here, we report that the chromatin modifier LSD1, an important regulator of AR transcriptional activity, undergoes epigenetic reprogramming in CRPC. LSD1 reprogramming in this setting activated a subset of cell-cycle genes, including CENPE, a centromere binding protein and mitotic kinesin. CENPE was regulated by the co-binding of LSD1 and AR to its promoter, which was associated with loss of RB1 in CRPC. Notably, genetic deletion or pharmacological inhibition of CENPE significantly decreases tumor growth. Our findings show how LSD1-mediated epigenetic reprogramming drives CRPC, and they offer a mechanistic rationale for its therapeutic targeting in this disease. Cancer Res; 77(20); 5479-90. ©2017 AACR.

Abstract A21: Characterization of the immune environment in the in situ to invasive breast carcinoma transition

Reactivation of immune responses against cancer cells—immunotherapy—is one of the few cancer therapies that can successfully eliminate even metastatic disease in a relatively nontoxic manner. However, its success has been limited to a subset of patients. For example, in breast cancer only ~20% of triple-negative breast cancer (TNBC) patients benefit from anti-PDL1 therapy. One reason for this limited success can be that different tumors evade the immune system via different mechanisms, which suggests that they may respond to different types of immunotherapies. Epithelial cancer cells in ductal carcinoma in situ (DCIS) are physically separated from the tumor-infiltrating leukocytes by the myoepithelial cell layer and the basement membrane, whereas in invasive ductal carcinoma (IDC), the epithelial cancer cells are intermingled with leukocytes. Therefore, we hypothesize that the DCIS to IDC transition is a key step in tumor progression as cancer cells are under different selection pressures, and only those that can evade the immune system can continue tumor progression, hence shaping subsequent tumor evolution. To dissect the role of leukocytes in the DCIS to IDC transition, we began by analyzing the composition and molecular profiles of leukocytes, with special emphasis on T cells, in normal breast tissues, DCIS, and IDC. We found that the relative frequency of leukocytes increases during tumor progression but the CD8/CD4 T cell ratio decreases. In addition, the gene expression profile of CD45+CD3+ T cells is different in DCIS compared to those isolated from normal breast tissue and IDCs. We found that gene set signatures corresponding to CD8+ T cells and NKT cells were enriched over regulatory T-cell signatures in DCIS compared to IDC. This result suggested that DCIS had a more activated immune environment compared to IDC. We further examined T-cell activation by immunofluorescence (IF) analysis and found a higher percentage of activated GZMB+CD8+ T cells in DCIS compared to IDC including a set of matched DCIS and locally recurrent IDC. We also found that the TCR clonotype was more diverse in DCIS than in IDCs. Interestingly, we detected a few relatively frequent clones that were shared among different DCIS patients, one of which was previously shown to recognize a protein from the Epstein-Bar virus. In order to dissect mechanisms of immune evasion in IDC, we analyzed immune checkpoint genes and proteins by FISH and IF. We found that TIGIT+ T cells were slightly more frequent in DCIS than in IDC. In triple-negative IDC, there was high expression of PD-L1 in epithelial cells and in 3/10 cases amplification of CD274 (encoding PD-L1), whereas DCIS had lower expression of PD-L1 and no amplification of CD274. To further elucidate mechanisms of immune evasion, we explored the significance of a cluster of genes encoding several chemokines that are located in close proximity of ERBB2 (encoding HER2). When analyzing the HER2+ samples from the TCGA, we found that coamplification of the 17q12 chemokine cluster (CC) with ERBB2 was enriched in HER2+ER+ luminal-like tumors, whereas there was either no gain or loss of the cluster in the HER2+ER breast tumors. Interestingly, we found higher expression of both T-cell activation and exhaustion-related genes in tumors that lack CC gain. Moreover, when assessing a cohort of HER2+ samples by multicolor FISH and IF, we found an inverse correlation between CC amplification and activation of CD8+ T cells. There was no correlation between CC amplification and recruitment of macrophages or myeloid-derived suppressor cells. Overall our results show coevolution of cancer cells and the immune microenvironment during tumor progression. Citation Format: Carlos R. Gil del Alcazar, SungJin Huh, Muhammad B. Ekram, Anne Trinh, Lin L. Liu, Francisco Beca, Zi Xiaoyuan, Misuk Kwak, Helga Bergholtz, Ying Su, Lina Ding, Hege G. Russnes, Andrea L. Richardson, Kirsten Babski, Elizabeth Min Hui Kim, Charles H. McDonnell, III, Jon Wagner, Ron Rowberry, Gordon J. Freeman, Deborah Dillon, Therese Sorlie, Lisa M. Coussens, Judy E. Garber, Rong Fan, Kristie Bobolis, D. Craig Allred, Joon Jeong, So Yeon Park, Franziska Michor, Kornelia Polyak. Characterization of the immune environment in the in situ to invasive breast carcinoma transition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A21.

Abstract B16: Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer

Abstracts: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WAnnTriple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. Cancer genome sequencing studies focusing on TNBC failed to identify novel recurrently mutated cancer-driving genes, obviating immediate opportunities for targeted therapeutic development. Here we have identified BET bromodomain inhibitors (BBDIs) as promising novel therapeutic agents in TNBC. Specifically, we have found that TNBC cells are significantly and preferentially growth inhibited by BBDIs (e.g., JQ1) with IC50s in the low nM range compared to luminal breast cancer cells. The growth of established xenografts derived from TNBC cell lines and primary patient samples were also efficiently inhibited by BBDIs treatment. BRD4, a target of BBDIs, is highly expressed in TNBCs compared to luminal cell lines and its downregulation using shRNAs inhibits TNBC cell growth in cell culture and induces tumor regression in vivo. In line with the role of BRD4 in transcription restart after G2/M, BBDI treatment prevented cell cycle re-entry, arrested TNBC cells in early G1, apoptosis, and induced luminal epithelial cell differentiation. Using integrated epigenomic analysis, we have identified the direct transcriptional targets of BBDI in TNBC. BBDI was found to efficiently displace chromatin-bound BRD4 in sensitive TNBC cells. This effect was more pronounced at genomic regions exhibiting elevated Bio-JQ1 binding by Chem-seq, implying strong association between BRD4 and JQ1 genomic binding patterns. By genome-wide study with ChIP-seq and RNA-seq, we have also found that selective disruption of super-enhancers associated genes by JQ1, leading to deregulation of coordinated transcriptional pathways involved in cell proliferation, invasion, and survival.nnThe sensitivity of TNBC and other tumor types to BET inhibition establishes a rationale for clinical investigation and a motivation to understand mechanisms of resistance. After selecting derivatives with acquired resistance to BET inhibition in previously sensitive TNBCs, we identified a unique mechanism of epigenomic resistance to this epigenetic therapy. Resistant cells remained dependent on BRD4. However, TNBC cells adapt to BET bromodomain inhibition by maintaining recruitment of unmutated BRD4 to super-enhancers in a bromodomain-independent manner. Differential super-enhancer analysis found a significant gain in the number of SEs in resistant cells, and a less pronounced loss of fewer SEs. The gain of Bio-JQ1 SEs was associated with gain of BRD4 binding to these genomic loci and also with increased transcription of the associated genes. Among the top gained super-enhancers in resistant cells was an upstream and intragenic region of H3K27ac enrichment at the BCL-xL locus. Notably, BCL-xL was among the few highly up-regulated genes in resistant cells by expression profiling. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 due to decreased activity of PP2A that we identified as a BRD4 phosphatase. We have defined that hyper-phosphorylation of BRD4 increases the recruitment of BRD4 to MED1 and contributes to BBDI resistance. To explore the translational relevance of our findings, we conducted synergy screens with JQ1 and molecules targeting BCL-XL (ABT737), a gained super-enhancer in resistant cells, and modulators of BRD4 phosphorylation, a CK2 inhibitor CX-4945, and PP2A activator perphenazine (PPZ). We observed significant synergy between JQ1 and ABT737, CX-4945, and also PPZ implying that these drug combinations will likely achieve even higher efficacy in TNBCs than BBDIs alone.nnTogether, these studies provide a rationale for BET inhibition in TNBC and argue for combination strategies to anticipate clinical drug resistance.nnCitation Format: Shaokun Shu, Charles Lin, Housheng Hansen He, Robert Witwicki, Justin Roberts, Doris Tabassum, Yi Liang, Muhammad Ekram, Ernest Doherty, Jonathan Brown, Hisham Mohammed, Clive DSantos, Michael McKeown, Christopher Ott, Jun Qi, Min Ni, Prakash Rao, Melissa Duarte, Shwu-Yuan Wu, Cheng-Ming Chiang, Richard Young, Jason Carroll, Henry Long, Myles Brown, Shirley X. Liu, Clifford Meyer, James Bradner, Kornelia Polyak. Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B16.