Biology subjects

Thakore, P. I.

Publications and source records attributed to Thakore, P. I..

4 recordsLinked to original sources

bioRxiv2020

Massively parallel phenotyping of variant impact in cancer with Perturb-seq reveals a shift in the spectrum of cell states induced by somatic mutations

Genome sequencing studies have identified millions of somatic variants in cancer, but their phenotypic impact remains challenging to predict. Current experimental approaches to distinguish between functionally impactful and neutral variants require customized phenotypic assays that often report on average effects, and are not easily scaled. Here, we develop a generalizable, high-dimensional, and scalable approach to functionally assess variant impact in single cells by pooled Perturb-seq. Specifically, we assessed the impact of 200 TP53 and KRAS variants in >300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison to orthogonal assays. Surprisingly, KRAS variants did not merely fit into discrete functional categories, but rather spanned a continuum of gain-of-function phenotypes driven by quantitative shifts in cell composition at the single cell level. We further discovered novel gain-of-function KRAS variants whose impact could not have been predicted solely by their occurrence in patient samples. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, which can be applied in cancer and other diseases driven by somatic or germline coding mutations.

genomics↗

bioRxiv2020

Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells

Recent technological advances have enabled massively parallel chromatin profiling with single-cell Assay for Transposase Accessible Chromatin by sequencing (scATAC-seq) in thousands of individual cells. Here, we extend these approaches and present ATAC with Select Antigen Profiling by sequencing, ASAP-seq, a tool to simultaneously profile accessible chromatin and protein levels in thousands of single cells. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA (mtDNA) for clonal tracking, thus concomitantly capturing three distinct modalities in single cells. Importantly, ASAP-seq uses a novel bridging approach that repurposes antibody:oligo conjugates designed for existing technologies that pair protein measurements with single cell RNA-seq. We demonstrate the utility of ASAP-seq by revealing coordinated and distinct changes in chromatin, RNA, and surface proteins during native hematopoietic differentiation, peripheral blood mononuclear cell stimulation, and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.

genomics↗

bioRxiv2020

Multi-modal pooled Perturb-CITE-Seq screens in patient models define novel mechanisms of cancer immune evasion

Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFN{gamma}-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFN{gamma} pathway, allowing us to distinguish it as a novel mechanism of immune resistance. We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.

genomics↗

bioRxiv2020

Cycling cancer persister cells arise from lineages with distinct transcriptional and metabolic programs

Non-genetic mechanisms have recently emerged as important drivers of therapy failure in cancer (Salgia and Kulkarni, 2018), where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment (Vallette et al., 2019). While most cancer persisters, like their bacterial counterparts, remain arrested in the presence of drug, a rare subset of cancer persisters can re-enter the cell cycle under constitutive drug treatment (Sharma et al., 2010). Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drug. Here, using time-lapse imaging, we found that cycling persisters emerge early in the course of treatment of EGFR-mutant lung cancer cells with the EGFR inhibitor osimertinib. To study this rare, transiently-resistant, proliferative persister population we developed Watermelon, a new high-complexity expressed barcode lentiviral library for simultaneous tracing each cells clonal origin, proliferative state, and transcriptional state. Analysis of Watermelon-transduced PC9 cells demonstrated that cycling and non-cycling persisters arise from different pre-existing cell lineages with distinct transcriptional and metabolic programs. The proliferative capacity of persisters is associated with an upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation in specific subpopulations of tumor cells. Mitigating oxidative stress or blocking metabolic reprograming significantly alters the fraction of cycling persister cells. In human tumors, programs associated with cycling persisters were induced in malignant cells in response to multiple tyrosine kinase inhibitors. The Watermelon system enabled the identification of rare persister lineages, that are preferentially poised through specific gene programs to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

cancer biology↗