Muscle atrophy is a prevalent condition in illness and aging. Identifying novel pathways that control muscle mass may lead to therapeutic advancement. We previously identified Nur77 as a transcriptional regulator of glycolysis in skeletal muscle. More recently, we showed that Nur77 expression also controls myofiber size in mice. It was unknown, however, whether Nur77’s regulation of muscle size begins during developmental myogenesis or only in adulthood. To determine the importance of Nur77 throughout muscle growth, we examined myofiber size at E18.5, 3 weeks postnatal age, and in young adult mice. Using the global Nur77-/- mice, we showed that Nur77 deficiency reduced myofiber size as early as E18.5. The reduction in myofiber size became more pronounced by 3 weeks of age. We observed comparable reduction in myofiber size in young myofiber-specific Nur77-knockout mice. These findings suggest that Nur77’s effect on muscle growth is intrinsic to its expression in differentiating myofibers, and not dependent on its expression in myogenic stem cells. To determine the importance of Nur77 expression in muscle accretion in mature mice, we generated an inducible-, muscle-specific, Nur77-deficient mouse model. We demonstrated that tamoxifen-induced deletion of Nur77 in 3-month-old mice reduced myofiber size. This change was accompanied by increased activity of Smad2 and FoxO3, two negative regulators of muscle mass. The role of Nur77 in muscle growth was further elaborated in the cardiotoxin-induced muscle regeneration model. Compared to wildtype mice, regenerated myofibers were smaller in Nur77-/- mice. However, when normalized to saline-injected muscle, the recovery of sarcoplasmic area was comparable between Nur77-/- and wildtype mice. These findings suggest that Nur77 deficiency compromises myofiber growth, but not the regenerative capacity of myogenic progenitor cells. Collectively, the findings presented here demonstrate Nur77 as an important regulator of muscle growth both during prenatal and postnatal myogenesis.

The quest continues for targeted therapies to reduce the morbidity of chemotherapy and to improve the response of resistant leukemia. Adhesion of acute lymphoblastic leukemia (ALL) cells to bone marrow stromal cells triggers intracellular signals that promote cell-adhesion-mediated drug resistance (CAM-DR). Idelalisib, an U.S. Food and Drug Administration (FDA)-approved PI3Kδ-specific inhibitor has been shown to be effective in CLL in down-regulating p-Akt and prolonging survival in combination with Rituximab; herein we explore the possibility of its use in B ALL and probe the mechanism of action. Primary B ALL in contact with OP9 stromal cells showed increased p-Aktser473. Idelalisib decreased p-Akt in patient samples of ALL with diverse genetic lesions. Addition of idelalisib to vincristine inhibited proliferation when compared to vincristine monotherapy in a subset of samples tested. Idelalisib inhibited ALL migration to SDF-1α in vitro and blocked homing of ALL cells to the bone marrow in vivo. This report tests PI3Kδ inhibitors in a more diverse group of ALL than has been previously reported and is the first published report of idelalisib inhibiting homing of ALL cells to bone marrow. Our data support further pre-clinical evaluation of idelalisib for the therapy of B ALL.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking cancer treatment that has produced remarkable clinical efficacy for hematopoietic malignancies, yet “on-target off-tumor” toxicity due to lack of target specificity limits the therapeutic potential of CAR-T cells in solid tumors. We are developing dual CAR-T cells composed of a canonical activator CAR (aCAR), intended to elicit efficacy in solid tumors, and an inhibitory CAR (iCAR) designed to efficiently inhibit the aCAR activity in normal tissue and vital organs. The iCAR and aCAR scFvs of this system bind distinct cell-surface antigens that are widely co-expressed on the normal epithelial from which solid tumors originate. Upon antigen binding, signal propagation through iCAR cytoplasmic domain (iDomain) counteracts aCAR induction of CAR T-cell activation and killing; thus protecting normal tissue. Dual CAR-T cells are activated and kill when exposed to tumor cells that have lost iCAR target expression due to chromosomal loss-of-heterozygosity (LOH) which is common in all cancers. iCAR-targeted LOH generates absolute and irreversible tumor specificity therefore tumor-specific overexpression of the iCAR or aCAR targets is not required. We have carried out a comprehensive screen using human T-cells to identify the most effective inhibitory iDomains derived from cell-surface receptors that naturally inhibit or moderate immune cell activation. The cell-based assay suite developed for this screen consisted of cell lines that co-express iCAR and aCAR target at clinically relevant levels and isogenic partners that mimic LOH through CRISPR editing of the iCAR target. The expression of dual CARs in T cells as biscistronic constructs introduced with lentiviral vectors was successful in a limited number of cases, and it was necessary to develop alternative methods of iCAR aCAR co-expression to complete the screen. Combining aCAR transduction with iCAR mRNA Electroporation (T-REP) proved to be a useful method to disentangle iDomain potency and expression level. The screen identified new iDomains that may expand the potential of iCAR technology in developing strategies to treat solid tumors without compromising efficacy for safety. Citation Format: David Bassan, Jason Yi, Neta Chaim, Nir Bujanover, Sarit Tabak, Tanya Kim, Yael Lopesco, Leehee Weinberger, Kristina Vucci, Michael Weist, Caitlin Schnair, Gregor B. Adams, Orit Foord, Frank J. Calzone, Rick Kendall, Adi Sharbi-Yunger. Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2848.

Translation of the unprecedented efficacy of chimeric antigen receptor (CAR) T-cell technology into solid tumors requires a solution to the problem of on-target damage of vital organs. We have developed an inhibitory CAR platform (iCAR) that imposes self-regulation in CAR T-cells to restrict inappropriate activation against normal cells without compromising tumor efficacy. The system consists of an iCAR that is co-expressed with a conventional activating CAR (aCAR) designed to activate T-cells. The iCAR and aCAR scFvs bind distinct cell-surface antigens that are ubiquitously expressed across all solid tumor histologies and normal tissues. The iCAR is allele-specific whereas the aCAR is pan-allelic. The iCAR technology was validated in functional assays and NSG models with cancer cell lines that mimic loss-of-heterozygosity (LOH) which is common in most human cancers due to increased chromosomal instability. In this context, loss of iCAR target expression due to LOH generates irreversible target specificity. Tumor-specific overexpression of aCAR and iCAR targets, a common barrier to the clinical translation of potent agents targeting the cell-surface proteins is not required. We have shown using in vitro and in vivo models that CAR T-cell activation and target killing are fully inhibited by dual iCAR + aCAR antigen engagement. This outcome validates the concept of iCAR-mediated protection of normal cells in vital organs. In contrast, tumors that mimic iCAR target LOH through CRISPR editing were fully eradicated with aCAR engagement alone. T-cell activation, proliferation, and cytotoxic activity after exposure to target cells lacking iCAR target antigen was found to be quantitatively indistinguishable from the results obtained with single input aCAR CAR T-cells. These results suggest that iCAR technology can enable treatment of cancer patients with solid tumors using CAR T-cells that maximize potency and efficacy by mitigating on-target CRS, neurotoxicity, and inevitable organ damage. The restriction of CAR T activation to the local tumor environment by iCAR-mediated self-regulation directly addresses a fundamental technology gap that cannot be solved by CAR T dosing or aCAR modification alone. Citation Format: Michael R. Weist, Adi Sharbi-Yunger, Jason Yi, Caitlin Schnair, David Bassan, Tanya Kim, Sarit Tabak, Yael Lopesco, Leehee Weinberger, Nir Bujanover, Neta Chaim, Kristina Vucci, Orit Foord, Frank J. Calzone, Rick Kendall, Gregor B. Adams. Rewiring CAR T-cells for absolute solid tumor specificity and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2826.