Sanam Shahid

Single-cell T cell receptor (TCR) repertoire sequencing enables simultaneous profiling of gene expression and TCR sequences, providing a coregistered measure of T cell phenotypic diversity and clonal dynamics. However, principled probabilistic frameworks for joint modeling of clonotype and phenotype have remained limited. Here we introduce TCRi, a hierarchical Bayesian information-theoretic framework that infers the joint probability distribution between genetic clonotype and transcriptional phenotype from paired single-cell transcriptomic and TCR sequencing data. TCRi treats phenotype assignment as a probabilistic inference problem: a variational autoencoder embeds single-cell expression into a structured latent space, and a hierarchical Bayesian model learns clonotype-specific phenotype distributions through principled information-sharing across clonotypes, patients and covariates. From these distributions, TCRi computes information-theoretic quantities: clonotypic entropy, phenotypic entropy, phenotypic flux and mutual information between clonotype and phenotype. When TCRi was applied to four real-world datasets comprising 167,118 CD8 T cells from patients with cancer treated with anti-PD-(L)1 immune checkpoint blockade, mutual information emerged as a robust discriminator of therapeutic response.

Engineered T and NK cell therapies have widely been used to treat hematologic malignancies and solid tumors, with promising clinical results. Current chimeric antigen receptor (CAR) T cell therapeutics are, however, associated with treatment-related adverse events such as cytokine release syndrome (CRS) and are prone to immunologic exhaustion. CAR-NK therapeutics, while not associated with CRS, have limited in vivo persistence. We now demonstrate that a population of NK-like TCRαβ ⁺ CD8 T cells (T _KLR ), identified and expanded ex vivo through its expression of the activating receptor NKG2C, can be transduced to express a second-generation CD19 CAR (1928z). These CAR+ T _KLR demonstrates superior tumor clearance and longer persistence compared to 1928z CAR ⁺ conventional CD8 T cells (T _Conv ) and 1928z CAR+ NK cells in mouse xenograft models. Moreover, CAR-modified T _KLR had decreased expression of PD-1, TIM-3, and LAG-3 and elicited significantly reduced CRS compared to CAR ⁺ CD8 T _Conv in mouse xenograft models. Similarly, NKG2C ⁺ T _KLR engineered with a TCR targeting the NY-ESO-1 antigen exhibited robust tumor control compared to TCR-engineered CD8 T _Conv in a mouse xenograft model. These data establish NKG2C+ T _KLR as a promising platform for cell engineering, and offer a safer, more durable alternative to conventional CAR-T and CAR-NK therapies.

Supplemental Figure 1. Systematic domain-focused CRISPR screens of GAPs and GEFs and transcriptomic profiling identify ARHGAP45 as a hematologic malignancy-biased dependency linked to distinct genetic and risk subgroups. Supplemental Figure 2. ARHGAP45 is a selective dependency in hematopoietic malignancies, but dispensable for normal CD34+ hematopoietic stem/progenitor cells (HSPCs) in vivo. Supplemental Figure 3. ARHGAP45 regulates cell cycle and constrains AML differentiation. Supplemental Figure 4. ARHGAP45 suppresses RhoA activity, and hyperactivation of RhoA suppresses AML growth. Supplemental Figure 5. Loss of ARHGAP45 sensitizes AML to CDC42 inhibition. Supplemental Figure 6. Activity of CAR-like TCRs against ARHGAP45 derived HA-1H antigen in myeloid malignancies. Supplemental Figure 7. In vivo efficacy of CAR-like TCRs against AML cell lines or patient-derived xenograft (PDX) expressing HA-1H.

GTPase-activating proteins (GAP) and guanine nucleotide exchange factors (GEF) play key roles in cancer development, but their large number and potential redundancy have limited systematic evaluation. In this study, we perform unbiased genetic screens to identify GAPs and GEFs with cancer- and lineage-specific requirements, as well as dual perturbation screens to dissect functionally relevant interactors of GAPs and GEFs. Application to primary specimens from patients with acute myeloid leukemia uncovers the GAP ARHGAP45 as a targetable dependency shared across cancers of hematopoietic origin while being dispensable in normal hematopoiesis. We demonstrate that targeting ARHGAP45-expressing cells can be achieved through T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-encoded minor histocompatibility antigen and that pharmacologic targeting of CDC42 required upon ARHGAP45 depletion augments ARHGAP45-directed cell therapies. These studies provide a resource for probing oncogenic and druggable regulators of GTPases and strategies to target a GAP that represents a shared dependency across blood cancers.Significance:In this study, we systematically interrogated GAPs and GEFs in cancer and identified the GAP ARHGAP45 as a dependency shared across blood cancers while dispensable in normal hematopoiesis. Targeting ARHGAP45-expressing cells is achievable via T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-derived antigen and small-molecule inhibition of GTPases required upon ARHGAP45 loss.

Supplementary Table S1. Clinical sample information. Supplementary Table S2. Domain-focused sgRNA libraries used in this study and MAGeCK analysis of CRISPR screening results. Supplementary Table S3. sgRNA sequences. Supplementary Table S4. Gene sets used in this study. SupplementaryTable S5. TMT-based total proteomics and phospho-proteomics.

GTPase-activating proteins (GAP) and guanine nucleotide exchange factors (GEF) play key roles in cancer development, but their large number and potential redundancy have limited systematic evaluation. In this study, we perform unbiased genetic screens to identify GAPs and GEFs with cancer- and lineage-specific requirements, as well as dual perturbation screens to dissect functionally relevant interactors of GAPs and GEFs. Application to primary specimens from patients with acute myeloid leukemia uncovers the GAP ARHGAP45 as a targetable dependency shared across cancers of hematopoietic origin while being dispensable in normal hematopoiesis. We demonstrate that targeting ARHGAP45-expressing cells can be achieved through T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-encoded minor histocompatibility antigen and that pharmacologic targeting of CDC42 required upon ARHGAP45 depletion augments ARHGAP45-directed cell therapies. These studies provide a resource for probing oncogenic and druggable regulators of GTPases and strategies to target a GAP that represents a shared dependency across blood cancers. Significance: In this study, we systematically interrogated GAPs and GEFs in cancer and identified the GAP ARHGAP45 as a dependency shared across blood cancers while dispensable in normal hematopoiesis. Targeting ARHGAP45-expressing cells is achievable via T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-derived antigen and small-molecule inhibition of GTPases required upon ARHGAP45 loss.

Despite clinical benefit with the use of chimeric antigen receptor (CAR) T cells, the need to manufacture patient-specific products limits its clinical utility. To overcome this barrier, we developed an allogeneic “off-the-shelf” CAR T-cell product using Epstein-Barr virus (EBV)–specific T cells (EBV-VSTs) genetically modified with a CD19-specific CAR (19-28z). Patients with relapsed/refractory (R/R) B-cell malignancies were stratified into 3 treatment cohorts: cohort 1 (n = 8; disease recurrence after allogeneic or autologous hematopoietic cell transplantation [HCT]), cohort 2 (n = 6; consolidative therapy after autologous HCT), or cohort 3 (n = 2; consolidative therapy after allogeneic HCT). The primary objective of this trial was to determine the safety of multiple CAR EBV-VST infusions. Most patients (n = 12/16) received multiple doses (overall median, 2.5 [range, 1-3]) with 3 × 106 T cells per kg determined to be the optimal dose enabling multiple treatments per manufactured cell line. Severe cytokine release syndrome or neurotoxicity did not occur after infusion, and no dose-limiting toxicity was observed in the trial. Median follow-up was 48 months (range, 4-135) with 4 deaths due to disease progression. Overall survival of all patients was 81% at 12 months and 75% at 36 months. Postinfusion expansion and persistence were limited, and CAR EBV-VSTs demonstrated a unique T-cell phenotype compared with autologous 19-28z CAR T cells. Our study demonstrates the feasibility and safety of an allogeneic “off-the-shelf” CAR EBV-VST product with favorable outcomes for patients with CD19+ R/R B-cell malignancies. This trial was registered at www.ClinicalTrials.gov as #NCT01430390.

Engineered T and NK cell therapies have widely been used to treat hematologic malignancies and solid tumors, with promising clinical results. Current chimeric antigen receptor (CAR) T cell therapeutics have, however, been associated with treatment-related adverse events such as cytokine release syndrome (CRS) and are prone to immunologic exhaustion. CAR-NK therapeutics, while not associated with CRS, have limited in vivo persistence. We now demonstrate that an NK-like TCRαβ ⁺ CD8 T cell subset, identified and expanded ex vivo through its expression of the activating receptor NKG2C (NKG2C ⁺ NK-like T cells), can be transduced to express a second-generation CD19 CAR (1928z), resulting in superior tumor clearance, longer persistence and decreased exhaustion compared to conventional 1928z CAR ⁺ CD8 T cells and 1928z CAR+ NK cells. Moreover, CAR-modified NKG2C ⁺ NK-like T cells resulted in significantly reduced CRS compared to conventional CAR ⁺ CD8 T cells. Similarly, NKG2C ⁺ NK-like T cells engineered with a TCR targeting the NY-ESO-1 antigen exhibit robust tumor control and minimal exhaustion compared to TCR-engineered conventional CD8 T cells. These data establish NKG2C ⁺ NK-like T cells as a robust platform for cell engineering, and offer a safer, more durable alternative to conventional CAR-T and CAR-NK therapies.