Stressed target cancer cells drive nongenetic reprogramming of CAR T cells and solid tumor microenvironment

Abstract The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumors is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquire early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogram and reverse the immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells of healthy donors or metastatic female breast cancer patients, induce robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a promising therapy for solid tumors

Supplementary Tables S1 to S5 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Table S1: Eligibility Criteria. Table S2: Genomic alterations within the PIK3CA pathway and other documented alterations for this study. Table S3: VAF of EOT alterations. Table S4: Comparison of Acquired PIK3CA mutations based on baseline activating mutation. Table S5: Comparison of Acquired PIK3CA mutations based on number of baseline activating PIK3CA mutations.</p

Supplementary Figures S1 to S11 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Supplementary Figure S1: PI3K pathway activity in selected cases with acquired PTEN alteration. Supplementary Figure S2. Validation of AKT constructs expression in T47D cells. Supplementary Figure S3. AKT activating mutations confer resistance to PI3Ka inhibitors. Supplementary Figure S4: Free energy calculations predict resistance to orthosteric PI3K inhibitors due to specific double PIK3CA mutants. Supplementary Figure S5: Free energy perturbation predicts reduced binding of orthosteric PI3K inhibitors to double mutants. Supplementary Figure S6. Expression of PIK3CA mutations in T47D cells. Supplementary Figure S7. MCF7 cells expressing W780R or Q859H double mutants show differential response to PIK3CA orthosteric inhibitors. Supplementary Figure S8: Chemical structure of RLY-2608. Supplementary Figure S9: Surface plasmon resonance (SPR) binding assay. Supplementary Figure S10. Alpelisib shows reduced potency of downstream signaling inhibition in the presence of W780R or Q859H/K. Supplementary Figure S11. T47D cells expressing I817F or E726K double mutants do not show a differential response to inavolisib (A) or RLY-2608 (B).</p

Supplementary Figures S1 to S11 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Supplementary Figure S1: PI3K pathway activity in selected cases with acquired PTEN alteration. Supplementary Figure S2. Validation of AKT constructs expression in T47D cells. Supplementary Figure S3. AKT activating mutations confer resistance to PI3Ka inhibitors. Supplementary Figure S4: Free energy calculations predict resistance to orthosteric PI3K inhibitors due to specific double PIK3CA mutants. Supplementary Figure S5: Free energy perturbation predicts reduced binding of orthosteric PI3K inhibitors to double mutants. Supplementary Figure S6. Expression of PIK3CA mutations in T47D cells. Supplementary Figure S7. MCF7 cells expressing W780R or Q859H double mutants show differential response to PIK3CA orthosteric inhibitors. Supplementary Figure S8: Chemical structure of RLY-2608. Supplementary Figure S9: Surface plasmon resonance (SPR) binding assay. Supplementary Figure S10. Alpelisib shows reduced potency of downstream signaling inhibition in the presence of W780R or Q859H/K. Supplementary Figure S11. T47D cells expressing I817F or E726K double mutants do not show a differential response to inavolisib (A) or RLY-2608 (B).</p

Supplementary Tables S1 to S5 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Table S1: Eligibility Criteria. Table S2: Genomic alterations within the PIK3CA pathway and other documented alterations for this study. Table S3: VAF of EOT alterations. Table S4: Comparison of Acquired PIK3CA mutations based on baseline activating mutation. Table S5: Comparison of Acquired PIK3CA mutations based on number of baseline activating PIK3CA mutations.</p