Biology subjects

Scandura, J. M.

Publications and source records attributed to Scandura, J. M..

4 recordsLinked to original sources

bioRxiv2026

CAR-T Targeting of Mutant Calreticulin Establishes a Potentially Curative Stem Cell-Directed Therapy for Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are sustained by mutated hematopoietic stem cells (HSCs). Existing therapies fail to eliminate this compartment, leaving allogeneic HSC transplantation as the only curative option. Recurrent MPN driver mutations in calreticulin (CALRmut) generate a C-terminal neopeptide that requires cell-surface expression for oncogenic signaling, making it an attractive immunologic target. However, it remains unknown if CALRmut is uniformly displayed on all MPN HSCs within hematopoietic microenvironments. We generated huAB2, a high-affinity CALRmut-specific humanized antibody, to use as the targeting domain for chimeric antigen receptor (CAR)-T cells. We show that CALRmut is consistently displayed on functional MPN HSCs and accessible in vivo. huAB2 CAR-T cells eradicate MPN-propagating CALRmut HSCs in patient-derived tumor xenograft models without antigen escape while preserving coexisting normal human and host hematopoiesis. These findings establish CALRmut display as an obligate feature of MPN HSC fitness and support the feasibility of curative, non-transplant immunotherapy for CALRmut MPNs. SignificanceTherapies that eradicate cancer stem cells enable cure, but their feasibility is unknown. We establish an approach to potentially cure MPNs by proving mutant calreticulin to be a MPN stem cell marker that can be targeted by CAR-T cells to selectively wipe out disease in preclinical models of human MPNs.

cancer biology↗

bioRxiv2022

PRC2 Inactivating Mutations in Cancer are Synthetic Lethal with DNMT1 Targeted Therapy via Enhanced Viral Mimicry

Polycomb Repressive Complex 2 (PRC2) establishes and maintains di- and tri-methylation at histone 3 at lysine 27 (H3K27me2/3) in the genome and plays oncogenic and tumor suppressor roles in context-dependent cancer pathogenesis. While there is clinical success of therapeutically targeting PRC2 core component, EZH2, in PRC2-dependent cancers (e.g., follicular lymphoma, epithelioid sarcoma), it remains an unmet therapeutic bottleneck in PRC2-inactivated cancer. Biallelic inactivating mutations in PRC2 core components are a hallmark feature of high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive subtype of sarcoma with poor prognosis and no effective targeted therapeutics. Using a custom RNAi-based drop out screen, we observed that PRC2-inactivation is synthetic lethal with DNA methyltransferase 1 (DNMT1) downregulation; we further observed that small molecule DNMT inhibitors (DNMTis) resulted in enhanced cytotoxicity and antitumor response in PRC2-loss cancer context in vitro and in vivo. Mechanistically, DNMTi-mediated de-repression of retrotransposons (e.g., endogenous retroviral elements (ERVs)/LTR, LINE, SINE) and gene targets is partly restricted by PRC2, which potentially contributes to limited therapeutic activity in PRC2-wild-type (wt) cancer context. In contrast, DNMTi treatment synergizes with PRC2 inactivation and cooperatively amplifies the expression of retrotransposons (e.g., ERV/LTR, LINE, SINE), and subsequent viral mimicry response that promotes robust cell death in part through PKR-dependent double stranded-RNA (dsRNA) sensing. Collectively, our observations posit DNA methylation as a safeguard against anti-tumorigenic cell fate decisions in the context of PRC2-inactivation to promote cancer pathogenesis. Further, they identified a novel targeted therapeutic strategy in PRC2-inactivated MPNST and delineated the PRC2-inactivated cancer context for future preclinical exploration and clinical investigation of DNMT1-targeted therapies in cancer. SIGNIFICANCEPRC2-inactivation drives oncogenesis in various cancers but therapeutically targeting PRC2-loss has remained challenging. Here we show that PRC2 inactivating mutations sets up a tumor context-specific liability for synthetic lethal interaction with genetic and therapeutic inhibition of DNMT1. DNMT1 inhibitor-induced cytotoxicity in PRC2-loss cancer context is accompanied by innate immune signaling signature through PKR-mediated sensing of endogenous retrotransposons. These observations posit a therapeutic window via direct anti-tumor effect by DNMT1 inhibitors in PRC2-loss cancers, and point to potentials to be combined with innovative immunotherapeutic strategies to capitalize on innate immune signaling activation.

cancer biology↗

bioRxiv2019

Megakaryocyte TGFβ1 Partitions Hematopoiesis into Immature Progenitor/Stem Cells and Maturing Precursors

Erythropoiesis is a multiweek program coupling massive proliferation with progressive cellular differentiation ultimately enabling a limited number of hematopoietic stem cells (HSCs) to yield millions of erythrocytes per second1. Erythropoietin (Epo) is essential for red blood cell (RBC) production but this cytokine acts well after irreversible commitment of hematopoietic progenitor cells (HPCs) to an erythroid fate. It is not known if terminal erythropoiesis is tethered to the pool of available immature hematopoietic stem and progenitor cells (HSPCs). We now report that megakaryocyte-derived TGF{beta}1 compartmentalizes hematopoiesis by coupling HPC numbers to production of mature erythrocytes. Genetic deletion of TGF{beta}1 specifically in megakaryocytes (TGF{beta}1{Delta}Mk/{Delta}Mk) increased functional HSPCs including committed erythroid progenitors, yet total bone marrow and spleen cellularity and peripheral blood cell counts were entirely normal. Instead, excess erythroid precursors underwent apoptosis, predominantly those erythroblasts expressing the Epo receptor (Epor) but not Kit. Despite there being no deficiency of plasma Epo in TGF{beta}1{Delta}Mk/{Delta}Mk mice, exogenous Epo rescued survival of excess erythroid precursors and triggered exuberant erythropoiesis. In contrast, exogenous TGF{beta}1 caused anemia and failed to rescue erythroid apoptosis despite its ability to restore downstream TGF{beta}-mediated Smad2/3 phosphorylation in HSPCs. Thus, megakaryocytic TGF{beta}1 regulates the size of the pool of immature HSPCs and in so doing, improves the efficiency of erythropoiesis by governing the feed of lineage-committed erythroid progenitors whose fate is decided by extramedullary renal Epo-producing cells sensing the need for new RBCs. Independent manipulation of distinct immature Epo-unresponsive HSPCs within the hematopoietic compartments offers a new strategy to overcome chronic anemias or possibly other cytopenias.

cell biology↗

bioRxiv2018

High throughput droplet single-cell Genotyping of Transcriptomes (GoT) reveals the cell identity dependency of the impact of somatic mutations

Defining the transcriptomic identity of clonally related malignant cells is challenging in the absence of cell surface markers that distinguish cancer clones from one another or from admixed non-neoplastic cells. While single-cell methods have been devised to capture both the transcriptome and genotype, these methods are not compatible with droplet-based single-cell transcriptomics, limiting their throughput. To overcome this limitation, we present single-cell Genotyping of Transcriptomes (GoT), which integrates cDNA genotyping with high-throughput droplet-based single-cell RNA-seq. We further demonstrate that multiplexed GoT can interrogate multiple genotypes for distinguishing subclonal transcriptomic identity. We apply GoT to 26,039 CD34+ cells across six patients with myeloid neoplasms, in which the complex process of hematopoiesis is corrupted by CALR-mutated stem and progenitor cells. We define high-resolution maps of malignant versus normal hematopoietic progenitors, and show that while mutant cells are comingled with wildtype cells throughout the hematopoietic progenitor landscape, their frequency increases with differentiation. We identify the unfolded protein response as a predominant outcome of CALR mutations, with significant cell identity dependency. Furthermore, we identify that CALR mutations lead to NF-{kappa}B pathway upregulation specifically in uncommitted early stem cells. Collectively, GoT provides high-throughput linkage of single-cell genotypes with transcriptomes and reveals that the transcriptional output of somatic mutations is heavily dependent on the native cell identity.

cancer biology↗