Biology subjects

Inghirami, G.

Publications and source records attributed to Inghirami, G..

9 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↗

bioRxiv2026

A patient derived xenograft repository capturing clinical and molecular heterogeneity of large B-cell lymphoma

Large B-cell lymphomas (LBCLs) are a clinically and molecularly diverse group of malignancies with a rapidly evolving therapeutic landscape that has introduced new areas of clinical need such as post-CD19 chimeric antigen receptor T (CART19) progression. Patient derived xenograft (PDX) models are an important tool for mechanistic studies and preclinical evaluation of new therapies and can be generated from a variety of clinical contexts that capture tumor-intrinsic resistance mechanisms. We therefore undertook a comprehensive effort to generate PDX models that encompass the molecular landscape of LBCLs and include important clinical scenarios for new drug development. Here we describe the first 48 models within this publicly available repository, capturing the transcriptional and genetic subsets of LBCL. These models also include 23 generated from post-CART progression biopsies which reproduce patterns of progression driven by CD19 mutation or expression loss, as well as tumor cell-intrinsic CART19 resistance that we validate in vivo. STATEMENT OF SIGNIFICANCEYang et al. describe X-LYMPH (Xenografts of Lymphoma), a publicly available and molecularly annotated PDX repository that captures the heterogeneity of large B-cell lymphoma. X-LYMPH includes models of chimeric antigen receptor T cell resistance, providing a shared foundation for mechanistic research and therapeutic development for lymphomas.

cancer biology↗

bioRxiv2025

Single-cell spatial mapping reveals dynamic bone marrow microarchitectural alterations and enhances clinical diagnostics in MDS

Myelodysplastic neoplasms (MDS) disrupt bone marrow hematopoiesis, yet clinical assessment relies largely on blast enumeration and qualitative morphology, which incompletely capture marrow architecture and disease state. We applied whole-slide multiplex immunofluorescence imaging with single-cell phenotyping to map bone marrow microarchitecture in MDS. Diagnostic biopsies (n=36), longitudinal treatment samples (n=29), precursor states (n=13), and normal controls (n=21) were analyzed, comprising >5 million spatially resolved cells. MDS marrow exhibited coordinated, genotype-imprinted architectural remodeling, including altered progenitor composition and spatial patterning, disrupted erythroid island organization, and displacement of hematopoietic stem and progenitor cells from perivascular niches. Interrogation of 82 cellular and spatial features yielded a composite Microarchitectural Perturbation Score (MDS-MAPS), derived from diagnostic samples and fixed prior to longitudinal analyses. In leave-one-patient-out cross-validation, MDS-MAPS discriminated remission from active disease more accurately than blast percentage (AUC 0.883 vs 0.660) and distinguished low-blast MDS from clonal cytopenia of undetermined significance (CCUS) (AUC 0.815). Mixed-effects modeling showed MAPS decreased in remission statistically independent of blast burden, with architectural normalization during remission and re-emergence at relapse. These findings define quantitative bone marrow architecture as a dynamic tissue-state biomarker that complements molecular and blast-based assessment in MDS.

pathology↗

bioRxiv2024

Mutability and hypermutation antagonize immunoglobulin codon optimality

The efficacy of polyclonal antibody responses is inherently linked to paratope diversity, as generated through V(D)J recombination and somatic hypermutation (SHM). These processes arose in early jawed vertebrates; however, little is known about how immunoglobulin diversity, mutability, and hypermutation have evolved in tandem with another more ubiquitous feature of protein-coding DNA - codon optimality. Here, we explore these relationships through analysis of germline IG genes, natural V(D)J repertoires, serum VH usage, and monoclonal antibody (mAb) expression, each through the lens of multiple optimality metrics. Strikingly, proteomic serum IgG sequencing showed that germline IGHV codon optimality positively correlated with VH representation after influenza vaccination, and in vitro, codon deoptimization of mAbs with synonymous amino acid sequences caused consistent expression loss. Germline V genes exhibit a range of codon optimality that is maintained by functionality, and inversely related to mutability. SHM caused a load-dependent deoptimization of IGH VDJ repertoires within human tonsils, bone marrow, and lymph nodes (including SARS-CoV-2-specific clones from mRNA vaccinees), influenza-infected mice, and zebrafish. Comparison of natural mutation profiles to true random suggests the presence of selective pressures that constrain deoptimization. These findings shed light on immunoglobulin evolution, providing unanticipated insights into the antagonistic relationship between variable region diversification, codon optimality, and antibody secretion; ultimately, the need for diversity takes precedence over that for the most efficient expression of the antibody repertoire.

immunology↗

bioRxiv2023

SINGLE CELL RESOLUTION SPATIAL MAPPING OF HUMAN HEMATOPOIESIS REVEALS AGING-ASSOCIATED TOPOGRAPHIC REMODELING

The spatial anatomy of hematopoiesis in bone marrow has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized bone marrow core biopsy tissue specimens, providing a rich resource for studying the intact human bone marrow topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence (AI)-based digital image analysis, and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group, and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and perivascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of inflammaging, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human bone marrow states using intact archival tissue specimens.

systems biology↗

bioRxiv2022

Whole genome error-corrected sequencing for sensitive circulating tumor DNA cancer monitoring

Circulating cell-free DNA (ccfDNA) sequencing for low-burden cancer monitoring is limited by sparsity of circulating tumor DNA (ctDNA), the abundance of genomic material within a plasma sample, and pre-analytical error rates due to library preparation, and sequencing errors. Sequencing costs have historically favored the development of deep targeted sequencing approaches for overcoming sparsity in ctDNA detection, but these techniques are limited by the abundance of ccfDNA in samples, which imposes a ceiling on the maximal depth of coverage in targeted panels. Whole genome sequencing (WGS) is an orthogonal approach to ctDNA detection that can overcome the low abundance of ccfDNA by supplanting sequencing depth with breadth, integrating signal across the entire tumor mutation landscape. However, the higher cost of WGS limits the practical depth of coverage and hinders broad adoption. Lower sequencing costs may thus allow for enhanced ctDNA cancer monitoring via WGS. We therefore applied emerging lower-cost WGS (Ultima Genomics, 1USD/Gb) to plasma samples at [~]120x coverage. Copy number and single nucleotide variation profiles were comparable between matched Ultima and Illumina datasets, and the deeper WGS coverage enabled ctDNA detection at the parts per million range. We further harnessed these lower sequencing costs to implement duplex error-corrected sequencing at the scale of the entire genome, demonstrating a [~]1,500x decrease in errors in the plasma of patient-derived xenograft mouse models, and error rates of [~]10-7 in patient plasma samples. We leveraged this highly de-noised plasma WGS to undertake cancer monitoring in the more challenging context of resectable melanoma without matched tumor sequencing. In this context, duplex-corrected WGS allowed us to harness known mutational signature patterns for disease monitoring without matched tumors, paving the way for de novo cancer monitoring.

genomics↗

bioRxiv2020

HSP90 facilitates oncogenic alterations of metabolism in B-cell lymphomas

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here, we described a novel role of HSP90 in the cytosolic compartmentalization of metabolic pathways in proliferating cancer cells. We found that HSP90 assists in the organization of metabolic enzymes into non-membrane-bound functional compartments termed metabosomes. Under experimental conditions that conserved the cellular proteostasis, we demonstrated that the compartmentalizing activity of HSP90 is critical to sustain the coordinated synthesis of multiple metabolites required for energy production, maintenance of the cellular biomass and secretion of immunometabolites. Conversely, inhibition of the nucleating capacity of HSP90 modified the topology of cytosolic metabosomes before protein degradation was apparent decreasing the efficiency of MYC-driven metabolic pathways. Inhibition of HSP90 decreases cancer metabolism in B-cell lymphoma cells and patients providing a novel mechanism of activity for this class of drugs.

cancer biology↗

bioRxiv2020

Unacylated ghrelin and AZP531 suppress the 3D growth of breast cancers

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, it remains the second leading cause of cancer-related death in the US. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. One major area of research relates to its reported effects to increase insulin sensitivity in diabetics, this being the basis for the development of unacylated ghrelin analog, AZP531 or livoletide, now in clinical trials for the treatment of Prader-Willi Syndrome. The mechanism of action of unacylated ghrelin is largely uncharacterized, in any system, because it does not bind to or activate the cognate ghrelin receptor, GHSR1a. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP531. We report potent effects of unacylated ghrelin, at picomolar doses, on the growth of breast cancer cells, dependent on 3D culture and activation of Gi. Suppression of MAPK signaling by unacylated ghrelin leads to cell cycle arrest and apoptosis. AZP531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

cancer biology↗

bioRxiv2019

Selective inhibition of HDAC3 targets synthetic vulnerabilities and activates immune surveillance in lymphoma

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6/HDAC3 onco-repressor complex. Accordingly, we show that HDAC3 selective inhibitors fully reverse CREBBP mutant aberrant epigenetic programming resulting in: a) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and b) restoration of immune surveillance due to induction of BCL6 repressed IFN pathway and antigen presentation genes. By reactivating these genes, exposure to HDAC3-i restored the ability of tumor infiltrating lymphocytes to kill DLBCL cells in an MHC II and MHC I dependent manner. Hence HDAC3-i represent a novel mechanism-based immune-epigenetic therapy for CREBBP mutant lymphomas. STATEMENT OF SIGNIFICANCEWe have leveraged the molecular characterization of different types of CREBBP mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in CREBBP mutant cells in tandem with promoting anti-tumor immunity.

cancer biology↗