Biology subjects

Behbehani, G. K.

Publications and source records attributed to Behbehani, G. K..

6 recordsLinked to original sources

bioRxiv2025

Allogeneic Polyclonal CD38KO/CD38-CAR γδT Cells For The Treatment Of T Cell Malignancies

Relapsed and refractory T cell malignancies are associated with poor clinical outcomes. Autologous sources of {beta}T cells have been employed for chimeric antigen receptor (CAR) therapies to eliminate the potential for graft-vs-host disease (GvHD). However, the application of CAR-T therapy for T-ALL has been hindered by an inability to obtain sufficient healthy {beta}T cells from patients combined with fratricide due to concurrent antigen expression on normal T cells. Here, we genetically engineered polyclonal {gamma}{delta}T cells, which do not cause GvHD, as an allogeneic source for cancer immunotherapy targeting the pancancer antigen CD38. Utilizing a novel expansion protocol in combination with CRISPR/AAV gene editing, we developed CD38KO/CD38-CAR polyclonal {gamma}{delta}T cells that target T-ALL. Our editing strategy enabled site-directed, on-target insertion of the CD38-CAR transgene into the CD38 locus, with no evidence of significant random CAR DNA integration (as commonly seen with lentiviral CAR transduction) or chromatin abnormalities resulting from CRISPR editing. This enhanced targeting effectively mitigated fratricide through simultaneous CD38 disruption and CAR expression. We demonstrated the efficacy of the CD38KO/CD38-CAR {gamma}{delta}T cells in vitro across multiple patient-derived T-ALL samples collected at baseline and relapse. In vivo, a single injection of CD38KO/CD38-CAR {gamma}{delta}T cells without exogenous cytokine support resulted in potent anti-leukemic efficacy. Fratricide-resistant CD38KO/CD38-CAR polyclonal {gamma}{delta}T cells thus represent a promising off-the-shelf therapeutic platform for T cell malignancies and other CD38-expressing cancers. Key PointsO_LIHybrid pan-{gamma}{delta}TCR antibody/mbIL21-41BBL feeder expansion yields high-purity, polyclonal {gamma}{delta}T cells suitable for CRISPR/AAV editing. C_LIO_LIOn-target CD38-CAR knock-in with simultaneous CD38 knockout prevents fratricide and enables potent T-ALL killing in vitro and in vivo. C_LI

immunology↗

bioRxiv2025

Natural Killer Cell Receptor Signaling and Activation Depend on Cell Cycle Stages

Receptor signaling in Natural Killer (NK) cells leads to post-translational modification (e.g., phosphorylation) of sub-cellular signaling proteins within minutes of receptor stimulation that eventually give rise to diverse effector functions including cell proliferation. Recent single-cell mass cytometry (i.e., CyTOF) experiments in macrophages showed variations of abundances of phosphorylated signaling proteins across cell cycle states indicating a dependence of cell signaling kinetics on an order of magnitude slower kinetics (~ several hours) of cell cycle transitions. We investigated cell cycle dependence of NKG2D signaling kinetics in NK cells by CyTOF measurements performed on IL-2-treated NKG2D-stimulated primary human CD56dim NK cells. The CyTOF experiments revealed monotonic or semi-monotonic increases of the average protein abundances of the majority of signaling proteins such as pCrkL, pPLC{gamma}2, and pErk, and the degranulation marker protein CD107a with progressing cell cycle states at specific time points post-NKG2D stimulation; however, several proteins such as pVav1, pS6, and pAkt, and early activation marker protein CD69 also showed non-monotonic variations in the average abundances with progressing cell cycle states. We used minimal mathematical and computational models coupling signaling and cell cycle processes to show that non-monotonic variations in the signaling protein abundances with progressing cell cycle stages are likely to arise in situations where protein synthesis and degradation and signaling kinetics are actively regulated by cell cycle processes.

immunology↗

bioRxiv2024

circPCMTD1: A protein-coding circular RNA that regulates DNA damage response in BCR/ABL-positive leukemias

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

cancer biology↗

bioRxiv2022

Differential correlation across subpopulations of single cells in subtypes of acute myeloid leukemia

Mass cytometers can record 40-50 parameters per single cell for millions of cells in a sample, and in particular, for leukemic cells. Many methods have been developed to cluster phenotypically similar cells within cytometry data, but there are fewer methods to visualize activity and interactions of pairs of proteins across these populations. We have developed a workflow for analyzing correlations associated with predfined populations. By clustering blood samples from acute myeloid leukemia (AML) patients and normal controls using an established algorithm, we obtained a minimum spanning tree of clusters of single cells. Using surface marker expression, we identified clusters on the tree that belonged to phenotypes of interest. Next, we computed correlations between pairs of proteins in each cluster. We developed a novel, coherent, probability-based statistic to test differences between vectors of correlation coefficients. By comparing all combinations of the normal controls under the statistic, we created an empirical distribution that provided a conservative measure of differential correlation. Using this empirically-derived distribution to define significance, we compared pooled samples from AML subtypes and normal controls to detect differential correlations. Given the structure present within this cytometry data set, we found it natural to consider correlations in this manner versus aggregating all data and computing a single correlation. Our results have the advantage that we can localize the statistical measure to determine contributions from particular phenotypic populations. Differentially correlated pairs of proteins can be further explored by considering a populations distribution of correlation coefficients or biaxially plotting protein expressions within individual cells in a given population. Our approach leads to a better understanding of the nonlinear relationships that exist in the cytometry data. Author summaryWe introduce a novel method for analyzing the abundance of single cell data collected by high-throughput technologies. Due to the high dimensionality of such datasets, there is a need for methods to identify significant interactions between genes or proteins. In particular, we are interested in statistical differences between correlations of proteins within populations of cells determined by traditional immunophenotyping techniques. In this paper, we have demonstrated the utility of this new framework in the case of blood samples from individuals with different subtypes of leukemia and compare to healthy controls. The motivation for this application is that differences between can illuminate potential drivers of the disease. We have illustrated an example of how intracellular events can be detected by our statistic. Finally, this method is flexible in that it can also be applied in a variety of contexts where there are vectors of correlation coefficients that need to be compared.

systems biology↗

bioRxiv2021

CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3{zeta} and NKG2D/2B4+CD3{zeta}) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

immunology↗

bioRxiv2019

Profiling Myelodysplastic Syndromes by Mass Cytometry Demonstrates Abnormal Progenitor Cell Phenotype and Differentiation

PurposeWe sought to enhance the cytometric analysis of MDS by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS.\n\nExperimental DesignTwenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering.\n\nResultsThis high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance (ICUS). These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells.\n\nConclusionsThese results demonstrate that high-parameter cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS.\n\nStatement of SignificanceO_LIHigh-dimensional mass cytometry enables high-resolution characterization of abnormal maker expression and myeloid development in MDS.\nC_LIO_LIThis technology could enhance MDS diagnosis and therapeutic monitoring and merits further research.\nC_LI\n\nStatement Translational RelevanceIn spite of several studies suggesting the utility of flow cytometry in the diagnosis of myelodysoplastic syndrome (MDS), this technique has not been widely adopted. We sought to enhance the utility of cytometry in MDS by performing the first high-dimensional mass cytometry characterization of a cohort of MDS patients. High-dimensional mass cytometry allowed all bone marrow cell populations to be simultaneously analyzed enabling high-resolution characterization of abnormal maker expression and myeloid development in MDS. This approach could identify almost all previously identified aberrant surface marker expression patterns in MDS while simultaneously enabling analysis by unsupervised clustering. Additionally, this mass cytometry analysis approach enabled the modeling of abnormal differentiation in MDS. This technology could enhance MDS diagnosis and therapeutic monitoring and merits further research.

cancer biology↗