Nadia Arang, Postdoctoral Fellow at UCSF in the laboratory of Nevan Krogan. "Pharmacological targeting of protein-protein interaction complexes underlying drug resistance in Triple Negative Breast Cancer (TNBC)". Proteins typically do not function alone, but rather in complexes with other proteins or molecules through physical or functional interactions. These interaction networks are highly dynamic and are under tight spatial and temporal control to enable cellular responses to extrinsic and intrinsic factors. Rewiring of these networks, leading to aberrant protein-protein interactions (PPIs) have been found to be associated with numerous human diseases including cancer, and a body of work has emerged suggesting that cancer-associated PPIs can drive responses to therapy. Hence, comprehensive mapping of protein interactomes at basal cell states, and in the context of cancer can shed light on mechanisms underlying cancer initiation, progression, and resistance to therapy. Together, profiling and targeting of aberrant PPIs can be used to inform novel therapeutic strategies for cancers with unmet clinical needs. Once such cancer, triple-negative breast cancer (TNBC), is an intractable and highly aggressive form of breast cancer that constitutes up to 20% of all cases and presents the poorest survival across of all breast cancer subtypes. TNBC is characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), commonly seen in other breast cancer subtypes5,6. As a result, there are no currently approved targeted therapies for TNBC and while standard chemotherapy is a standard treatment approach, drug resistance is commonly developed throughout the course of treatment5. Taken together, this underscores a critical gap in our understanding of the molecular mechanisms driving TNBC that could benefit from the application of systems biology approaches, and highlights an urgent need for novel therapeutic strategies for patients with TNBC. While a great deal of transcriptomic profiling has been performed on TNBC tumors to identify subtypes based on molecular characteristics, little is known about the protein interaction landscape and how this may contribute to tumor progression and resistance to therapy. I hypothesize that unique protein complexes may underlie the resistance of TNBC to therapy and that systematic mapping of the PPI landscape of TNBC will provide insight into the complex molecular mechanisms associated with disease that can be pharmacologically targeted to re-engage therapeutic responses. Towards this end, I propose profiling of the dynamic protein complexes in TNBC in the context of drug resistance and in response to therapy using size exclusion chromatography-mass spectrometry (SEQ-MS) paired with a targeted drug screen in order to develop a comprehensive systems-based understanding of the protein interaction networks that govern drug response in TNBC. Of note, the data generated through this proposal is well-poised to be validated in collaboration with the I-SPY 2 multicenter phase II clinical trial for high-risk early-stage BRCA patients based at UCSF. All I-SPY 2 patients are profiled for gene and protein/phosphoprotein expression, which presents a unique opportunity to test response predictors and resistance mechanisms in TNBC to improve patient survival.

Ronald Babu, Assistant Researcher at UCSF in the laboratory of Nevan Krogan. "Massively parallel base editing screen to unlock cancer susceptibility across the PI3K-AKT-mTOR pathway in breast cancer". Somatic mutations in PIK3CA has been linked to 40% incidence of breast cancer, which is second most common cancer among women in United States. Cancer genome sequencing has catalogued a list of PIK3CA gene variants (germline, somatic or VUS-variance of uncertain significance) yet failed to functionally map such variants to breast cancer development or any other phenotypic consequences. 20% of the PIK3CA mutations are missed by sequencing based diagnostic kits (Therasreen)2 emphasizing an urgent need to evaluate these undetected PIK3CA mutations. The main drivers of breast cancer progression are associated with mutations acquired in the PI3K-AKT-mTOR axis which either lead to gain or loss of function. Clinically lots of attention has been drawn towards developing drugs targeting PI3K-AKT-mTOR either upstream or downstream of the pathways, some of which are already approved my FDA. However, several studies emphasized on the acquired resistance to such drugs due to somatic mutations and the need to functionally map the specific roles played by such mutations. Here I propose to perform a multi-modal massively parallel base editing screen for systematic mapping of loss of function (LOF)/gain of function (GOF) variants in PIK3CA-Akt1-mTOR pathway. Mutagenesis of the top-scoring genes (PIK3CA, Akt1, mTOR, PTEN) will be performed using cytidine base editors (CBEs) and adenine base editors (ABEs) for mapping the burden of each variant along with VUS associated with cancer progression. As acquired mutations are core to development of drug resistance in cancer, our base editing screen will enable us to endogenously scan the effect of gene alteration at the nucleotide level.

Kimberly Badal, Postdoctoral Fellow at UCSF in the laboratory of Laura Esserman at UCSF. "Elucidating the epigenomic alterations associated with toxic environmental chemical exposures and breast cancer risk in the Athena Breast Health Network using machine learning". Epidemiological studies have linked several toxic environmental chemical exposures to an increased breast cancer risk. Cell and animal studies have shown that some of these chemicals promote breast cancer development though the epigenetic regulation of gene expression. Historically, the field of exposure estimation has been limited to measuring one, known chemical at a time to assess the association with breast cancer risk. Presently, in collaboration with the UCSF Environmental Research and Translation for Health (EaRTH) Centre, we are utilizing a new non-targeted chemical screening assay, which can screen for 3800 industrial chemicals in the blood. We are assessing the chemical body burden of 100 women in the Athena Breast Health Network in a pilot case-control 1:1 study using blood samples taken at least one year before diagnosis. To complement this data, we propose to characterize the methylome of these 100 women to investigate whether there are differences in the DNA methylation (DNAm) patterns of women with high vs. low chemical exposures. We will examine individual chemicals and chemical mixtures which is critical given that women are simultaneously exposed to multiple chemicals throughout their life course. To the best of our knowledge, this will be the first study to pair non-targeted chemical screening data with DNAm data to understand breast cancer risk. We will then leverage a new deep learning tool to discover DNAm sites associated with exposures and then investigate the relationship between the sites found and breast cancer risk. This project aligns with the CCMI goal to go beyond DNA to understand the molecular drivers of cancer as it will provide two additional layers of information – chemical exposures and DNA methylation. This project will benefit from CCMI expertise in understanding how the DNAm patterns found may operate to drive breast cancer development. In the long-term, if reliable DNAm biomarkers of historical exposure to chemicals that increase breast cancer risk can be found, it could be used to tailor prevention and screening interventions for women at risk. This may either reduce the incidence of breast cancer or find disease earlier for women at high risk, with the goal of reducing breast cancer mortality.

Verima Pereira, Postdoctoral Fellow at UCSF in the laboratory of John Gordan. "A Physical and Genetic Interaction Map to Predict the Liver Cancer Response to VEGFR2 Inhibition". Liver cancer or Hepatocellular carcinoma (HCC) is a leading cause of cancer death in the US and worldwide. HCC produces high levels of vascular endothelial growth factor (VEGF) and angiopoietin 2 (ANGPT2) resulting in disorganized vasculature and establishing an "angiogenic immune checkpoint" which hinders anticancer immunity. Current treatments target VEGF and VEGFR2 (VEGF receptor 2) to improve responses to anti-cancer immune therapy, but no treatments are available targeting ANGPT2. Thus, ANGPT2 is a key player in patient outcomes. Importantly, ANGPT2 is transcriptionally controlled by VEGFR. I have found that ANGPT2 levels are altered following VEGFR2 inhibition in HCC cell lines via effects on nuclear factor of activated T cells (NFAT) transcription factors (Fig 1B). Strikingly, while VEGFR2 inhibition decreases ANGPT2 expression in some HCC lines, it increases it in others. This may mirror effects in patients, with decreased ANGPT2 further enabling an immune response, while increased ANGPT2 could drive resistance. Thus, decoding tumor cell responses to VEGFR2 inhibition could allow us to predict if a patient will benefit or not from the current standard of care treatments for HCC. I propose leveraging affinity purification-mass spectrometry (AP-MS) and CRISPRi techniques to decode intricate molecular associations governing anti-VEGF therapy responses concerning ANGPT2, using HCC cell lines as a model. This innovative approach aims to augment understanding and facilitate enhanced prediction of ANGPT2-related responses, as depicted visually.

Sami Norreddine, Postdoctoral Fellow at UC San Diego in the laboratory of Prashant Mali. "Unlocking BRAF-Targeted Therapy Resistance through Functional Mapping of Protein Interaction Domains". Protein-protein interactions play a crucial role in oncogenic signaling and the regulation of cell growth and survival. In human cancers the most frequent alterations occur on MAPK pathway mainly via BRAF and RAS activating mutations. Inhibitors targeting the MAPK pathway have been developed, including BRAF inhibitorslike vemurafenib and dabrafenib, as well as MEK1/2 inhibitors such as trametinib. Initially approved for melanoma treatment, their use has recently been expanded as tissue-agnostic therapy for adult and pediatric solid tumors with the presence of a BRAF activating mutation (BRAFV600). This approval represents a significant progress for many cancers with BRAF mutations such as non-small lung cancer, biliary tract cancer, glioma, hairy cell leukemia, thyroid carcinoma, and many other rare cancers. Despite demonstrating efficacy in many cancer types, resistance to MAPK inhibitors often develops over time and represent a major challenge. One of the common mechanisms of acquired resistance are RAS mutations which upregulate alternative signaling pathways such as PI3K/AKT. Understanding the specific protein-protein interactions involved in these pathways is crucial for developing new therapeutic strategies and overcome drug resistance. Recently, Dr. Mali’s laboratory has developed a novel screening methodology called Peptide-Tiling (PepTile), whereby lentiviral libraries of peptides comprehensively tiling proteins of interest are assayed via pooled fitness screens to identify protein domains essential for cell fitness. This method has enabled identification of protein domains and peptides with antiproliferative properties in breast cancer and melanoma cell lines. Melanoma has been at the forefront of targeted therapy development, including MAPK inhibitors. Many lessons learned from melanoma studies have informed the development of therapies for other cancers. Insights gained from melanoma resistance mechanisms can potentially be translated to improve treatments for other malignancies. Thus, we propose to perform a comprehensive screening of peptides using PepTile to identify novel targets for the treatment of MAPK resistant BRAF-driven melanomas by revealing essential protein domains involved in oncogenic signaling.

Alexandra Tankka, Graduate Student at UC San Diego in the laboratory of Gene Yeo, in collaboration with Trey Ideker. Characterization of RBP splicing activity that is essential for triple negative breast cancer survival. RBPs as fundamental regulators of oncogenesis. Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype lacking expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) [1]. Due to the absence of hormone receptor expression, TNBCs lack targeted therapies, emphasizing an urgent need for the discovery of effective targets. Accumulating evidence shows that TNBC RNA expression profiles driven by RNA-binding proteins (RBPs) are significantly different from that in hormone receptor-positive BC tumors and cells originating from normal tissues. Additionally, RBPs have recently emerged as fundamental regulators of oncogenesis due to their aberrant expression in tumor tissues which can lead to cancer-driving RNA modifications. Despite these findings, RBPs are broadly unexplored as TNBC drug targets because their systematic characterization is limited by a lack of functional and scalable assays for investigating of RBP function. Cancer-associated splicing dysregulation promotes triple negative breast cancer progression. Alternative splicing plays a significant role in cancer, leading to alterations in relative isoform expression of particular mRNAs which can result in the loss- or gain-of-function of key tumor suppressors and oncogenes.The dynamic regulation of these alternative splicing events is caused by the dysregulation of splicing regulatory RBPs, or splicing factors (SFs). For instance, the SF3B1 core spliceosomal component plays a key role in the survival and proliferation of TNBC, however, pharmacological modulators of the SF3B subcomplex disrupt early stages of spliceosome assembly and lead to toxic effects in normal tissues, limiting its utility as a safe therapeutic. Thus, identifying splicing regulatory RBPs required for cancer cell progression but dispensable for normal cell survival can serve as a more efficient therapeutic strategy. TRA2B was shown to cooperate with the MYC oncogene to promote the proliferation of TNBC cells while having no significant effect on normal cells. Furthermore, depletion of BUD31 leads to global defects in splicing patterns due to the enhanced pressure on the spliceosome in TNBC compared to normal mammary cells. This suggests that disruption of specific splicing regulatory RBPs non-essential for spliceosome assembly in normal cells is a key vulnerability in TNBC. Therefore, I hypothesize that there are uncharacterized splicing regulatory RBPs only required for TNBC survival and their specific downstream splicing events can be exploited to inhibit growth and survival. Since individual proteins rarely mediate cellular behaviors, I propose to employ a systems biology approach to reveal TNBC-specific splicing regulatory networks required for tumor development using large-scale functional studies.

Pham Vo, Graduate Student at UC San Diego in the laboratory of Silvio Gutkind, in collaboration with Emma Lundberg. "Signaling network controls transition from quiescence to growth competence". Cell proliferation plays crucial roles in a wide range of biological activities, including growth and development, wound healing, cancer, and immune response. This process is energetically costly and must be tightly co-regulated to generate specific cellular outcomes across molecular, cellular and tissue scales. However, the growing list of molecular parts that participate in these processes represents a major challenge to our ability to predict, design and control cellular behavior. In collaboration with Dr.Emma Lundberg (CCMI 2.0 faculty), who is a leader in spatiotemporal proteins mapping through the integration of imaging data with transcriptomics and proteomics, we aim to perform a systematic interrogation of the signaling events and genotype-phenotype relationships that give rise to “quiescence to growth competence” transition. This is complemented by analysis of transcriptomic, proteomic, and epigenetic changes. As a model system, I will use hTERT-RPE1 epithelial cells, which are non-cancerous cells known to enter in a quiescent state upon growth factor deprivation and can be induced to re- initiate cell proliferation by mitogenic stimulation. We hypothesize that mitogenic signaling networks initiate the transition from quiescence to proliferation through a highly orchestrated transcriptional and epigenetic response. By exploring the signaling circuitry controlling the transition from quiescence to growth competence and understanding how this process is different between normal cells and cancer cells, our long-term goal is to uncover how cancer cells override controlling mechanisms restraining their unrestricted capacity by mapping spatiotemporal protein variations associated with cell proliferation in normal cells and tumor cells. Ultimately, this knowledge will provide insights to identify novel therapeutic targets for cancers. This fits into the CCMI mission of generating comprehensive maps of the key protein-protein interactions underlying cancer, which can be translated to identify new drug targets. The hands-on experience, scientific and technical expertise that I will gain from this proposed work will prepare me for the transition to become a cancer biology investigator that employs systems biology approaches to discover new paradigms that can translated into the clinic in the future.