Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the western world. Cancer evolution is a major obstacle to CLL treatment, as clonal evolution enables malignant cells to diversify and evade therapy. In the setting of chemoimmunotherapy, CLL’s ability to adapt is directly linked to intra-tumoral heterogeneity, and tumor evolution following therapy is the rule rather than the exception. Recently, ibrutinib targeted therapy has dramatically altered the therapeutic landscape of CLL, achieving high response rates even in patients with high-risk disease. Yet, despite its clinical efficacy, disease progression is increasingly appreciated, with mutations in BTK and PLCG2 identified as genetic adaptations to therapy. Intriguingly, some patients who progress on ibrutinib do not exhibit known resistance genotypes. The first part of our project is focused on discovery interrogating additional potential genetic mechanisms of resistance to BTK inhibition via whole genome sequencing of pre-treatment and relapse samples from patients that lack a substantial allelic burden of known resistance mutations. Moreover, some relapse samples exhibit a low frequency of cells with known resistance mutations, suggesting that inter-clonal collaboration may drive the resurgence of wildtype cells comingled with ibrutinib resistance mutations cells. In the second part of our project, we will study this phenomenon, utilizing Genotyping of Transcriptomes (GoT), a novel strategy developed by the Landau lab to amplify a locus within a gene of interest using a small part of the cDNA generated by 10x single-cell RNAseq platform. We will apply this technology to genotype an admixture of wildtype and mutant cells, enabling direct linkage of genotypes to scRNAseq profiles. High-resolution mapping of CLL subsets will identify transcriptional programs that are associated with persistence and escape from therapy, which may ultimately suggest novel strategies for subsequent combination therapy.
mTORC1 is the major player in the evolutionarily conserved signaling pathway responsible for regulating cellular homeostasis, which functions at the epicenter of the cells’ response to nutrient availability, and controls protein synthesis, metabolism and cell growth accordingly. Dysfunctional regulation of this complex has been associated with cancer and various inflammatory and neurological disorders. A kinase, mTOR is the drug target of rapamycin-analog therapies and is used to treat Tuberous Sclerosis Complex (TSC) patients where loss of its negative regulator TSC1 or TSC2 leads to uncontrolled mTOR activation and aberrant tumor growth, even under nutrient deficiency. We propose that metabolite-mTOR interplay is a key to answering how mTOR remains active under energetic stress in TSC. The primary goal of my project is to investigate the molecular mechanisms mediating mTORC1 activation based on biochemical enzymatic characterization aided by computational studies of structural effects. These studies will shed light on the activation mechanism of mTOR as well as the development of novel therapeutic strategies aimed at inhibiting metabolite-driven mTOR activity for the treatment of metabolically deregulated diseases.
Prostate cancer (PC) is the most commonly diagnosed malignancy and the second leading cause of cancer death among males. Treatment resistance is the number one cause of death in metastatic PC patients, while the molecular mechanisms underlying clinical drug resistance remain poorly elucidated. Circulating tumor cells (CTCs), represent a readily available, repeatedly accessible “liquid biopsy” source representative of the entire tumor burden, allowing interrogation of the molecular characteristics of the disease in real-time, in order to identify molecular drivers of disease progression. My project aims to investigate molecular determinants of clinical drug response/resistance through RNA-Seq analysis of CTCs derived from PC patients. CTC samples of this project are collected from PC patients in PROPHECY (Prospective CiRculating prOstate Cancer Predictors in HighEr Risk mCRPC studY) clinical trial. This trial represents a first-in-field comprehensive analysis of CTC molecular profiles for the development of a CTC molecular taxonomy of mCRPC. Currently, we are analyzing RNA-Seq data of CTCs collected from peripheral blood of 40 PC patients before treatment (Baseline) and after treatment with the next-generation Androgen Receptor signaling inhibitors, abiraterone or enzalutamide (Progression 1). Ultimately, we want to identify clinically actionable genes/pathways and somatic mutations underlying drug resistance in metastatic PC patients.
Loss of imprinting (LOI) of the Dlk1-Dio3 gene cluster has been reported in 50-75% of Acute Myeloid Leukemia (AML) patients but the underlying mechanisms remain elusive. DNA methylome datasets from more than 2000 AML patients, as well as ERRBS data from (pre)leukemic mouse models, show aberrant DNA methylation at the Dlk1-Dio3 locus. LOI results in aberrant DNA methylation of differentially methylated regions (DMRs) and may thus contribute to leukemic transformation. In the past year, we dissected how the imprinting control Region (called IG-DMR) and the somatic Gtl2 DMR control imprinting of Dlk1-Dio3. Using an allele-specific reporter system, we found that the IG-DMR consists of two antagonizing regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which maintains expression of maternal genes by precluding de novo methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression-, DNA methylation- and 3D chromatin topology patterns. Although, targeted epigenetic repression of either DMR is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the active DNA (de)methylation machinery. Ultimately, we aim to delineate the factors that protect the DMRs from de novo DNA methylation/demethylation, which could lead to potential new diagnostic or therapeutic targets for AML.
Diffuse large B-cell lymphomas (DLBCL) is the most common type of non-Hodgkin lymphoma in the United States and worldwide. Two genomic studies revealed that 8-16% of DLBCLs harbor loss-of-function mutations in the gene TOX (TOX^LOF). TOX is a transcription factor that regulates lymphoid tissue development. Interestingly, TOX^LOF events co-occur with mutations that result in constitutive activation of NF-kB pathway. NF-kB is a family of transcription factors that control the expression of genes involved in survival, proliferation, stress responses and inflammation. These associations suggest a potential onco-suppressive role for TOX in DLBCL, especially in the context of NF-kB activation. However, whether a mechanistic link between the activity of TOX and NF-kB exist is not known, nor it is known how TOX^LOF modifies the biology of NF-kB-driven lymphomas. The overall goal of my project is to elucidate the molecular mechanism(s) by which lack of TOX expression sustains NF-kB lymphomagenesis. Understanding the biology of NF-kB lymphomas is of high clinical relevance, as it will allow identifying novel therapeutic opportunities toward a subgroup of aggressive DLBCLs for which no curative therapy is currently available.
The kidney is a common site for metastases arising from malignant cells associated with a variety of primary lesions including the skin, lung, breast, stomach, and pancreas. However, once cancer cells intravasate and enter the blood stream, it is unknown what factors promote specific extravasation at the kidney over other possible metastatic sites. We propose that cooperation between tumor cells in the blood stream and endothelial cells in the kidney vasculature promote such specific targeting. We thus sought to understand tumor-extrinsic mechanisms promoting cancer cell extravasation through the vasculature at characteristic end organs by comparing the transcriptional profiles of endothelial cells associated with the kidney, heart, lung, and liver. Due to extreme functional heterogeneity within the kidney vasculature, we also performed single cell RNA-sequencing to determine transcriptional networks associated with different functional zones in the renal vasculature. Differential expression analysis revealed a variety of kidney-specific transcriptional networks, including several factors specifically expressed in the glomerular endothelial cell population that may have potential to affect transmigration through endothelial cells. We screened candidates by over-expressing candidate genes in HUVEC cells and co-culturing with lung and breast cancer cell lines. The T-Box3 (Tbx3) transcription factor stood out as a protein that promoted a dramatic increase in attachment to the endothelial cell populations when over-expressed. We are currently investigating the regulatory network downstream of Tbx3 that may contribute to increased endothelial cell affinity. Collectively, this study will be the first to reveal a cooperation between metastatic cancer cells and vasculature of the kidney to promote extravasation and metastatic colonization.
Prostate cancer remains the most commonly diagnosed cancer and the second-leading cause of cancer-related death among American men. Despite recent advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of prostate cancer, acquired resistance ultimately ensues. A significant subset of patients with resistant disease develops AR-null, androgen signaling-indifferent prostate tumors that lose their luminal identity and progress to neuroendocrine prostate cancer (NEPC). We previously identified MYCN (which encodes the transcription factor N-Myc) as a driver of NEPC. While the ability of N-Myc to downregulate AR signaling and promote the development of NEPC has been well-characterized, the function of N-Myc and the molecular program that drives continued tumorigenesis in the absence of androgen remain unknown. We have developed several human and mouse in vitro and in vivo N-Myc prostate cancer cell line models and our preliminary data demonstrate that N-Myc overexpression results in castration-resistant tumor growth. Intriguingly, RNA-seq analyses revealed dramatically different N-Myc target gene expression signatures between castrated and non-castrated recipients. My project is focused on understanding the interactions between N-Myc and its chromatin-bound co-factors to uncover novel therapeutic options for patients with N-Myc+ castration-resistant disease and NEPC.
Chronic anemias are a major medical problem with therapeutic options limited to blood transfusions and erythroid stimulating agents (ESAs). Unfortunately transfusions and ESAs are expensive, time consuming and very often ineffective. New approaches for treating chronic anemias are needed. It takes about 3 weeks for immature hematopoietic stem and progenitor cells to differentiate into red blood cells (RBCs). There is a lot known about the final 3 cell divisions leading to RBCs. Surprisingly little is known about how these terminal stages of erythropoiesis are matched by bone marrow production of more immature erythroid progenitor cells. My project is identifying how production of red blood cell progenitors in the bone marrow is coupled to terminal erythroid maturation. My work promises new therapies for chronic anemias to increase the outflow of immature progenitors with ESAs to support erythroid maturation.
Urothelial carcinoma (UC) is the fifth most commonly diagnosed cancer and is treated primarily with Platinum based chemotherapy. Previous research in our laboratory identified that APOBEC3 mutational signatures are enriched in chemotherapy-resistant UC (Faltas et. al Nat genetics, 2016). Building upon these observations, my research project focuses on understanding the effects of APOBEC3-induced mutations on the evolution of chemotherapy and immunotherapy resistance. By studying cells expressing the APOBEC3 gene family, which is thought to induce mutations in chemotherapy resistant UC, I want to understand how tumors develop resistance and how the immune system responds to the mutational changes driven by APOBEC3. My specific project wants to identify the role of APOBEC3-induced mutagenesis in cell fitness. I want to study how APOBEC3-induced mutagenesis gives bladder cancer cells a survival advantage by performing a competition assay between cells expressing APOBEC3 in an inducible system and those that do not. By combining cell imaging and mathematical modeling I want to measure the interaction between these two populations to explain their behavior.
My project seeks to investigate the mechanisms underlying transcriptional activity of the androgen receptor (AR) splice variant AR-v7 which is clinically correlated with poor prognosis in castrate resistant prostate cancer (CRPC) patients. AR signaling is a key driver of prostate cancer (PC) growth and metastatic progression. Thus, androgen deprivation therapy is the first line of treatment for PC. However, most patients develop castration resistant prostate cancer (CRPC), partly due to the expression of transcriptionally active AR splice variants (AR-Vs). AR-v7 is the most prevalent variant expressed in about 60% of CRPC tumors. Currently, there is no therapeutic modality that can inhibit AR-v7 expression or activity and the exact mechanism by which transcription is activated by AR-v7 is unknown. My recent study revealed a unique AR-v7 intranuclear mobility mechanism suggesting that AR-v7 achieves its transcriptional activity despite short residence time on chromatin, which is distinct from AR-fl. We are preparing the manuscript to report this finding. To further develop specific therapeutic strategies to target AR-v7 in CRPC, we have developed cell-based assays which enable us to monitor nuclear translocation of AR-v7. Currently, we are performing small molecule high throughput screening (HTS) to identify compounds with the inhibitory effect on AR-v7 nuclear import. Successful completion of our drug discovery will lead the development of novel therapies to inhibit AR-v7 nuclear activity in CRPC.
My project aims to identify molecular mediators underlying T-cell dysfunction in non-small cell lung cancer (NSCLC). T-cells are key agents of the anti-tumor immune response, and enhancing their function through therapies such as anti-PD-1 has been shown to yield significant survival benefits for cancer patients. However, these benefits are temporary, and only effective in a subset of patients. We have previously shown that utilizing anti-PD-1 therapy in a mouse model of NSCLC yielded similar outcomes as observed in patients, with robust and cell-specific effects on T cell subsets driving this benefit (Markowitz et al, JCI Insight 2018). We have globally examined gene expression changes in tumor-infiltrating T-cells both in the presence and absence of anti-PD-1 therapy, and identified a cohort of >100 genes with significantly enhanced expression in the dysfunctional state both in mouse and human samples. We are utilizing antigen-specific in vitro and in vivo assays in mouse models to mechanistically dissect the relevance and potency of these dysregulated genes in modulating tumor progression. We further are developing ex vivo analysis platforms for examining and experimentally manipulating patient-derived tissue samples to explore relevance, mechanistic congruity, and therapeutic potential of our findings in the mouse models. Ultimately, this work will identify targets for potential future therapeutics, as single agents or in combination with anti-PD-1, to further enhance the anti-tumor immune response and improve outcomes for NSCLC patients.
Kras is the most frequently mutated oncogene, and specific cancer types show a clear bias in the types and frequency of Kras alterations. My project seeks to better understand the impact of the Kras mutational landscape through the generation and analysis of new genetically engineered mouse models (GEMMs). I have developed a series of new conditional Kras mouse models that recapitulate common Kras alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C). On my first year as a T32 trainee I was able to characterized how these different mutations in Kras drive remarkably differences in early stages of pancreatic cell transformation, associated with dramatic changes in the degree of stromal expansion within the pancreas. During this second year I have been granted I am focused in exploring how such changes following Kras alterations influence progression to carcinoma, dictate the degree and type of immune cell involvement in these tumors, and mediate the efficacy of immune checkpoint inhibitors. To do so I am currently generating pancreatic ductal adenocarcinoma (PDAC) models where a mutation in the tumor suppressor Trp53 it is created by CRISPR mediated base-editing technology. This cutting-edge approach has revolutionized the way to generate accurate mouse models that better reflect the genetics seen in cancer patients. Our lab has been a pioneer in the field by using and further optimizing the tool; thus, allowing us to publish this past July an article (Zafra et al, Nat Biotech, “Optimized base editors enable efficient editing in cells, organoids and mice”).