Biology subjects

Weinstock, D. M.

Publications and source records attributed to Weinstock, D. M..

10 recordsLinked to original sources

bioRxiv2025

Integrating Single-Cell Biophysical and Transcriptomic Features to Resolve Functional Heterogeneity in Mantle Cell Lymphoma

Intra-tumor heterogeneity impacts disease progression and therapeutic resistance but remains poorly characterized by conventional histologic, immunophenotypic, and molecular approaches. Single-cell biophysical properties distinguish functional phenotypes complementary to these approaches, providing additional insight into cellular diversity. Here we link both buoyant mass and stiffness to gene expression to identify clinically relevant phenotypes within primary mantle cell lymphoma (MCL) cells, employing MCL as a model of biological and clinical diversity in human cancer. Linked measurements reveal that buoyant mass and stiffness characterize B-cell development states from naive to plasma cell and correlate with expression of oncogenic B-cell receptor signaling genes such as BLK and CD79A. Additionally, changes in cell buoyant mass within primary patient specimens ex vivo correlate with sensitivity to Brutons Tyrosine Kinase inhibitors in vivo in MCL and chronic lymphocytic leukemia, another B-cell malignancy. These findings highlight the value of biophysical properties as biomarkers of response in pursuit of future precision therapeutic strategies.

cancer biology↗

bioRxiv2024

Mutation and cell state compatibility is required and targetable in Ph+ acute lymphoblastic leukemia minimal residual disease

Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples. HIGHLIGHTSO_LIRelapse after remission on TKI can harbor mutations in ABL1, RAS, or neither C_LIO_LIMutations and development-like cell state dictate fitness in residual disease C_LIO_LICo-targeting cell state and ABL1 markedly reduces MRD C_LIO_LIBiophysical measurements provide an integrative, rapid measurement of cell state C_LI

cancer biology↗

bioRxiv2023

Leukemia circulation kinetics revealed through blood exchange method

Leukemias and their bone marrow microenvironment are known to undergo dynamic changes over the course of disease. However, relatively little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of leukemia cell dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1-2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells.

cancer biology↗

bioRxiv2022

Targeting TRIP13 in Wilms Tumor with Nuclear Export Inhibitors

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes (XPO1 and KPNB1) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP13 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

cancer biology↗

bioRxiv2021

A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia

A small set of lineage-restricted transcription factors (TFs), termed core regulatory circuitry (CRC), control cell identity and malignant transformation. Here, we integrated gene dependency, chromatin architecture and TF perturbation datasets to characterize 31 core TFs in acute myeloid leukemia (AML). Contrary to a widely accepted model, we detected a modular CRC structure with hierarchically organized, partially redundant and only sparsely interconnected modules of core TFs controlling distinct genetic programs. Rapid TF degradation followed by measurement of genome-wide transcription rates revealed that core TFs directly regulate dramatically fewer genes than previously assumed. Leukemias carrying KMT2A (MLL) rearrangements depend on the IRF8/MEF2 axis to directly enforce expression of the key oncogenes MYC, HOXA9 and BCL2. Our datasets provide an evolving model of CRC organization in human cells, and a resource for further inquiries into and therapeutic targeting of aberrant transcriptional circuits in cancer.

genomics↗

bioRxiv2021

Micro-region transcriptomics of fixed human tissue using Pick-Seq

Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology and disease. Recently developed spatial transcriptomic methods use fresh-frozen specimens but most diagnostic specimens, clinical trials, and tissue archives rely on formaldehyde-fixed tissue. Here we describe the Pick-Seq method for deep spatial transcriptional profiling of fixed tissue. Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. We demonstrate the use of Pick-Seq with several different fixed and frozen human specimens. Application of Pick-Seq to a human melanoma with complex histology reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immuno-editing. Parallel imaging confirms changes in immuno-phenotypes and cancer cell states. This work demonstrates the ability of Pick-Seq to generate deep spatial transcriptomic data from fixed and archival tissue with multiplexed imaging in parallel.

cancer biology↗

bioRxiv2020

Sex-biased ZRSR2 mutations in myeloid malignancies impair plasmacytoid dendritic cell activation and apoptosis

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDCs). BPDCN occurs at least three times more frequently in men than women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. STATEMENT OF SIGNIFICANCESex bias in cancer is well recognized but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.

cancer biology↗

bioRxiv2020

Signaling input from divergent pathways subverts malignant B-cell transformation

Malignant transformation typically involves multiple genetic lesions whose combined activity gives rise to cancer1. Our analysis of 1,148 patient-derived B-cell leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway characteristic for the differentiation status of these transformed B cells. Specifically, we show here the JAK/STAT5 signaling pathway supports the developmental stage-specific expansion of pro-B ALL whereas the ERK-pathway that of pre-B ALL. Mutations that were not aligned with the central oncogenic driver would activate divergent pathways and subvert malignant transformation. Oncogenic lesions in B-ALL frequently mimic survival and proliferation signals downstream of cytokine receptors (through activation of STAT5)2-7 or the pre-B cell receptor (through activation of ERK)8-13. STAT5- (372 cases) and ERK- (386 cases) activating lesions were frequently found but only co-occurred in [~]3% (37) of cases (P=2.2E-16). Single-cell mutation and phosphoprotein analyses revealed that even in these rare cases, oncogenic STAT5- or ERK-activation were mutually exclusive and segregated to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed malignant transformation. Conversely, Cre-mediated deletion of divergent pathway components triggered leukemia-initiation and accelerated development of fatal disease. Thus, persistence of divergent signaling pathways represents a powerful barrier to malignant transformation while convergence on one principal driver defines a key event during leukemia-initiation. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits strongly synergized with direct inhibition of the principal oncogenic driver. Hence, pharmacological reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses and to overcome drug-resistance. Current treatment approaches for drug-resistant cancer are focused on drug-combinations to suppress the central oncogenic driver and multiple alternative pathways14-17. Here, we introduce a concept based on inhibition of the principal driver combined with pharmacological reactivation of divergent pathways.

cancer biology↗

bioRxiv2020

RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

BackgroundDown syndrome acute lymphoblastic leukemia (DS-ALL) is characterized by the high frequency of CRLF2-rearrangements, JAK2-mutations, or RAS-pathway mutations. Intriguingly, JAK2 and RAS mutations are mutually exclusive in leukemic sub-clones, causing dichotomy in therapeutic target choices. ResultsHere we show that in primary leukemic cells from DS-ALL, in the absence of RAS-mutations, wild-type (wt)RAS is active, and/or can be induced by the physiological ligand TSLP of the transmembrane-receptor CRLF2. We show active/inducible RAS in 14/20 (70%) of primary DS-ALL samples analyzed, 8 of which had no RAS-mutations, but 75% of those had either mutated or hyperphosphorylated JAK2. No wtRAS cases with mutated/hyperphosphorylated JAK2 were observed that lacked activated RAS protein. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. We show that TSLP boosts the direct binding of active PTPN11 to wtRAS. Pre-inhibition of RAS or PTPN11, but not of PI3K or JAK signaling, prevented TSLP-induced RAS-GTP boost. Using multivariate-clustering based on RAS-activity/inducibility we show significant separation between standard-risk and high-risk DS-ALL groups. Cox proportional-hazards model showed protein-activity (but not mutation status) as independently predictive of outcome. ConclusionsOur data indicate that RAS protein activity levels (and not JAK2/RAS mutation profiles), are predictive of outcome. Importantly, our data suggest that inhibition of RAS and direct RAS-pathway components should be combined with PI3K/mTOR and/or JAK2 inhibitors for high-risk cases. Therapeutically this is relevant for >75% of DS-ALL and our additional data suggest that it warrants further investigation in high-risk non-DS-ALL.

cancer biology↗

bioRxiv2018

Parp3 promotes long-range end-joining in murine cells

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classical- and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class switch recombination in primary B cells and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting Parp1 may suppress DSB processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

cancer biology↗