Rameen Beroukhim, M.D. Ph.D. - US grants

DESCRIPTION (provided by applicant): Summary: As a physician with a PhD in structural biology, I aim to become an independent investigator studying the somatic genetics of prostate cancer while maintaining a small clinical practice in oncology. I am an Instructor in Oncology at the Dana-Farber Cancer Institute, receiving training in the laboratory of Dr. Matthew Meyerson, with additional access to the resources of the Broad Institute of Harvard and MIT. In addition to coursework, conference attendance, and mentoring by Dr. Meyerson and others, I propose a research project to characterize chromosomal aberrations in prostate cancer, relate them to cancer progression, and identify targeted oncogenes and tumor suppressor genes. Prostate cancer is distinguished by its clinical heterogeneity, with some cancers remaining indolent and others requiring local therapy to prevent metastasis and death. Unfortunately, we do not know the genetic bases of these differences. Understanding this may allow development of markers to distinguish between aggressive and indolent prostate cancer, and therapeutics to target the molecular alterations that give rise to progressive disease. We have developed techniques to characterize chromosomal aberrations in prostate cancer, including loss of heterozygosity (LOH) and copy number changes, at high resolution and throughout the genome, using single nucleotide polymorphism (SNP) arrays. We are able to identify known oncogene and tumor suppressor gene targets with high accuracy, and have small and intriguing candidate gene lists for other targeted regions. Here we propose to use this technology to address the somatic genetics of prostate cancer progression and validate those gene targets, with three specific aims: 1, Identify chromosomal aberrations, including LOH and copy number alterations, differing in prevalence between localized and metastatic prostate cancers;2, Identify point mutations and insertion/deletion events in candidate oncogenes and tumor suppressor genes that appear targeted by these aberrations;and 3, Functionally validate selected candidate oncogenes from Aims 1 and 2. Relevance: Prostate cancer arises due to the accumulation of mutations in the DNA of a cell. We aim to identify mutations associated with its growth and spread outside of the prostate. Identifying them will potentially help us recognize in advance cancers that are likely to spread and develop therapies for them.

? DESCRIPTION (provided by applicant): Glioblastomas (GBMs) are genomically well characterized, yet heterogeneous, and exhibit profound resistance to all existing treatment strategies. The most effective therapeutics are radiation therapy (RT) and the alkylating agent temozolomide (TMZ), but progression typically occurs within months after initiating these treatments. The mechanisms underlying this profound resistance remain unknown, but genetic heterogeneity is likely a major contributor, as has been shown in other cancers. Unfortunately, little is known about how GBM genomes evolve with treatment. This information would be useful to guide development of strategies to avoid the development of resistance and to identify optimal therapeutic approaches in the recurrent setting. We hypothesize that somatic genetic profiles of GBMs that recur after treatment with RT and TMZ differ substantially from pre-treatment GBMs, and that the differences point to mechanisms by which GBMs resist these treatments. To test this, we propose to identify and functionally validate recurrent genetic changes associated with resistance using innovative genomic analysis tools and patient derived model systems. Our collaborative consortium has collected an unprecedented number of paired pre- and post-treatment human tumors (>200). We have also created more than 100 patient derived GBM models that will be treated to test for the emergence of recurrent resistance drivers. Preliminary data from both patient samples and models indicate substantial tumor evolution occurs during treatment and identify TP53, CHEK2 and other rational targets as candidate mediators of resistance. Collective analysis of the data will be used to address two Aims. In Aim 1, we will test the hypothesis that treatment with radiation and temozolomide leads to consistent genetic changes in human tumors using whole exome sequencing of paired pre- and post-treatment tumor samples to determine large-scale changes in population structures and single cell sequencing to evaluate the effects of these treatments on microheterogeneity. In Aim 2, we will test the hypothesis that genetic changes identified in post-treatment GBMs functionally contribute to RT and TMZ resistance in GBM using patient derived cell lines (PDCL) and patient derived xenografts (PDX). We will determine the effects of radiation and temozolomide on these models and their genomic hierarchies using deep sequencing and test the effects of candidate drivers of resistance both in vitro and in vivo. We will determine whether resistant clones exist prior to treatment or are stochastically induced using an innovative single cell barcoding approach to determine whether the evolution of clonal substructures is consistent across replicate experiments. These studies will create a comprehensive understanding of genetic evolution during standard-of-care therapy for GBM. They will inform diagnostic approaches for assignment of targeted therapeutics in the recurrent setting and identify genetic changes driving resistance. Therapeutic targeting of these novel resistance drivers could represent a rational approach to substantially improve our existing standard of care for GBM patients.

Abstract Somatic copy number alterations (SCNAs) are a type of mutation in cancer that affect more of the cancer genome than any other genetic event. SCNAs often contribute to cancer development and progression, and detecting them can contribute to the development of diagnostic and therapeutic advances in clinical care. As part of The Cancer Genome Atlas (TCGA) project our group characterized SCNAs for over 10,000 tumors across 30 different tumor types. Through these efforts we developed state-of-the-art methods to detect and interpret SCNAs, and used these to discover SCNAs that recur across many tumors and likely contribute to the formation of these tumors, the candidate tumor suppressors and oncogenes these SCNAs target, and novel clinically relevant SCNA-based cancer subtypes. We have also developed methods to detect SCNAs and the rearrangements that bound them from high-throughput sequencing data of the type being collected by the Genomics Data Analysis Network (GDAN). These methods resolve SCNAs, the mechanisms by which they arise, and their potential biological consequences, in much greater detail than could be done with microarray data generated for TCGA. Leveraging our experience in SCNA analysis, we propose to establish a Genomics Data Analysis Center (GDAC) that will service the GDAN with comprehensive, advanced analyses of SCNAs and the rearrangements that bound them, with the goals of identifying biologically and clinically relevant patterns of SCNA and disseminating this information to the GDAN and wider research community. We will: 1) Generate basic and quality control information for each tumor. We determine the fraction of cancer cells within each tumor (tumor purity) and the average copy number genomewide (ploidy). We will also test every putative pair of tumor and normal DNA samples to ensure that they did originate in the same person. 2) Characterize SCNAs and rearrangements in each tumor, including clonal and subclonal amplifications, deletions, loss of heterozygosity, and complex events like chromothripsis, firestorms, and isochromosomes. 3) Identify recurrent SCNAs and rearrangements that are likely to drive tumor development and progression, and the oncogenes and tumor suppressor genes they likely target. 4) Classify tumors by previously identified SCNA subtypes and discover new subtypes. We will identify SCNAs and genomewide patterns of SCNA that correlate with clinical and molecular features of tumors. 5) Integrate with the GDAN and research community. We will integrate our analytic pipelines with those of other GDACs; immerse ourselves in cooperative Analysis Working Groups formed by the GDAN to refine those analyses in light of the most important questions; make our analysis results available to other members of the GDAN in real time; and disseminate those results to the wider research community through our existing web portal and by working closely with other GDACs to integrate our analyses into their web portals. Our results will inform how SCNAs cause cancer and indicate new diagnostic and therapeutic strategies.

Abstract Children diagnosed with Diffuse Intrinsic Pontine Gliomas (DIPGs) are faced with a mortality rate of 100%. The current standard of care is radiation therapy. Despite achieving initial responses, tumors quickly exhibit resistance and start to grow again. We have taken leading positions in a national trial, DIPG-BATs, that has evaluated routine biopsy of DIPGs in children. We seek to take advantage of the tissue obtained through the DIPG-BATs trial to understand the genetic underpinnings of DIPG and their impact on therapeutic response. We also seek to identify therapeutic combinations that are sufficient to prevent the acquisition of radiation resistance. In Aim 1, we propose to analyze the largest set of DIPG whole genomes to date. We will combine sequencing data collected on the DIPG-BATs trial with those from newly diagnosed patients, in addition to previously published genomes. In Aim 2, we will evaluate the role of PPM1D mutations in generating resistance to radiation therapy. Our initial analysis of BAT biopsies has revealed that over 50% of DIPG genomes contain mutations in either PPM1D or TP53, and that these mutations are mutually exclusive. PPM1D has been characterized as a negative regulator of TP53, a critical facilitator of radiation sensitivity. In Aim 3, we will use both hypothesis- based and unbiased approaches to identify therapeutic combinations with radiation that increase efficacy. We will utilize genetic and pharmacological methods of inhibiting PPM1D and MDM2 in combination with radiation in patient-derived DIPG lines with TP53 mutations. We will also perform a genome-wide CRISPR-cas9 screen to identify genes whose suppression selectively increase radiation response in DIPG-relevant cell lines. These experiments will address at least three central questions regarding resistance to radiation therapy in DIPG: characterization of driver genomic alterations and identification of those that confer resistance to radiation therapy, determination of how alterations in p53 signaling confer resistance to radiation, and evaluation of PPM1D as a novel therapeutic target.

ABSTRACT Pediatric low-grade astrocytomas (PLGAs) as a group are the most common solid tumor in children. While rarely fatal, patients frequently experience a relapsing/remitting course wherein repeated cycles of chemotherapy or radiation are required to contain the disease, often inducing irreparable neurologic damage. Therefore, less toxic therapies are urgently needed. We and others have identified genetic drivers of PLGAs in order to develop more specific and effective targeted therapeutic strategies. This proposal addresses the second most common set of alterations in PLGAs, those involving MYB transcription factors (TFs). In recent studies we identified rearrangements involving MYB and MYBL1 in 10% of PLGAs. Each rearrangement is associated with distinct PLGA subtypes. Angiocentric gliomas harbor fusions between a truncated MYB and a truncated portion of the tumor suppressor and myelination gene, QKI. A second tumor subtype, diffuse astrocytoma, frequently exhibits MYBL1 truncation as the only oncogenic change. Similar genetic rearrangements of MYB/MYBL1 have now been implicated in leukemias and adenoid cystic carcinomas. While rearrangements in MYB/MYBL1 are common driver events in PLGAs the biological consequences of these oncogenic changes are not understood. To develop new therapeutic approaches we will determine how the distinct mutant MYB transcription factors contribute to oncogenesis, and whether altered QKI function is critical for growth of angiocentric gliomas. We will address the biology of MYB/MYBL1 oncogenes and implications for new therapies in the following Specific Aims: Aim 1: Test the hypothesis that MYB and MYBL1 alterations contribute to tumorigenesis via distinct but related mechanisms using ChIP-Seq and RNA-Seq to define transcription factor and signaling networks activated by MYB genes. Aim 2: Test the hypothesis that the MYB fusion partner QKI contributes to tumorigenesis by altering RNA processing. We will determine whether MYB- QKI retains RNA binding ability and thereby alters RNA splicing. Aim 3: Identify MYB activated genes functionally critical for tumor growth and amenable to targeting with small molecule therapeutics. This will be done by evaluating novel therapeutics to target MYB/MYBL1 protein regulation and using a CRISPR-screen to identify key gene dependencies for MYB-induced tumors. These studies will provide basic biological insights into the function of MYB proteins and QKI in pediatric astrocytomas and other cancers with MYB alterations. This knowledge will directly inform the development of new rational therapeutics for pediatric astrocytomas and other cancers with MYB transcription factor alterations.

Project Abstract Despite decades of research into targeted therapeutics, the most effective treatments in glioma remain DNA damaging agents: radiation and the alkylating agents temozolomide and nitrosureas like CCNU. In this project?s prior cycle, we found that mismatch repair deficiency (MMRd) is a common source of temozolomide resistance; and that unlike other cancers, gliomas that gain temozolomide resistance through MMRd tend not to respond to immune checkpoint inhibition. But they often do respond to CCNU. We hypothesize that a fuller understanding of the different resistance mechanisms to TMZ and CCNU will enable 1) improved knowledge of when and how to use these agents, including clinically useful biomarkers, and 2) optimization of combined strategies using targeted and immunotherapies developed over the last decade. Although extensive work has been done to understand how CCNU damages DNA and to detect genes and pathways involved in repairing this damage, the field lacks a unified understanding of how CCNU effects vary across gliomas with different DNA damage response (DDR) characteristics, how resistance arises, and how the effects of CCNU interact with other agents including DNA damaging agents such as temozolomide and radiation, as well as therapeutics targeting specific DDR functions and pathways. As a result, we lack biomarkers that can accurately guide clinicians to prescribe CCNU to patients who are likely to respond, do not know the optimal combined therapeutic approaches involving CCNU, and clinical practice varies widely. We propose to pursue a systematic evaluation of the genomic effects and potential therapeutic roles of CCNU. A major innovation in our proposal is our systematic approach to evaluating the effects of CCNU on cancer survival and proliferation and genome integrity: when used alone and in combination with temozolomide, RT, and agents targeting DNA damage response pathways; and across a wide variety of DNA damage response contexts. For this, we will leverage a living tissue biobank of over 250 gliomas in vivo and in vitro models and state-of-the-art technologies for functional genomics and genome characterization across treatment conditions and DDR backgrounds. Our Aims are: Aim 1: Test the hypothesis that MMRd based resistance to TMZ within a GBM indicates relative sensitivity to CCNU and RT and can be detected through plasma cell-free DNA. Aim 2: Test the hypothesis that defects in proteins involved in repair of CCNU-induced ICLs determine resistance to CCNU and strategies to overcome. Aim 3: Test the hypothesis that intentional manipulation of mutational profiles and clonal dynamics by coordinating TMZ, CCNU, RT, and DDR pathway inhibition can increase the effectiveness of immunotherapy. DNA damaging agents remain the most effective agents in glioma and all other cancers, the unified understanding of their effects in isolation and combination across the varied DDR contexts in this proposal will shape the use of these agents in clinical practice and guide the development of new biomarker-driven combinations with novel DDR targets.