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The actin cytoskeleton exists in a dynamic equilibrium with monomeric and filamentous states of its subunit protein actin. The spatial and temporal regulation of actin dynamics is critical to the many functions of actin. Actin levels are remarkably constant, suggesting that cells have evolved to function within a narrow range of actin concentrations. Here we report the results of screens in which we have increased actin levels in strains deleted for the ~4800 nonessential yeast genes using a technical advance called selective ploidy ablation. We detected 83 synthetic dosage interactions with actin, 78 resulted in reduced growth, whereas in 5 cases overexpression of actin suppressed the growth defects caused by the deleted genes. The genes were highly enriched in several classes, including transfer RNA wobble uridine modification, chromosome stability and segregation, cell growth, and cell division. We show that actin overexpression sequesters a limited pool of eEF1A, a bifunctional protein involved in aminoacyl-transfer RNA recruitment to the ribosome and actin filament cross-linking. Surprisingly, the largest class of genes is involved in chromosome stability and segregation. We show that actin mutants have chromosome segregation defects, suggesting a possible role in chromosome structure and function. Monomeric actin is a core component of the INO80 and SWR chromatin remodeling complexes and the NuA4 histone modification complex, and our results suggest these complexes may be sensitive to actin stoichiometry. We propose that the resulting effects on chromatin structure can lead to synergistic effects on chromosome stability in strains lacking genes important for chromosome maintenance.

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Background: Chimeric antigen receptor T cell therapy (CAR T) has demonstrated remarkable clinical efficacy in hematological malignancies. However, compromised T cell effector function, proliferation, and persistence can limit CAR T from reaching their full curative potential. Interleukin-2 (IL-2) is a potent stimulator of T cells, however therapeutic use of IL-2 is limited by systemic toxicity due its pleiotropy. Therefore, to provide a selective IL-2 signal to engineered T cells, we have developed a human orthogonal ligand/receptor system consisting of a half-life extended pegylated IL-2 mutein (STK-009) and a mutated IL-2 Receptor Beta (hoRb) that responds to STK-009 but not wild type IL-2. SYNCAR-001 is an autologous CAR T co-expressing a CD19 CAR and hoRb on the T cell surface. In mouse models of refractory lymphoma, STK-009 treatment led to expansion and activation of SYNCAR-001 cells with a maintenance of stem cell memory and effector T cell phenotypes. When added to SYNCAR-001, STK-009 increased complete response rate and durable responses in a dose dependent manner. In non-human primate studies, STK-009 alone demonstrated no significant biological activity in IL-2 sensitive populations (T cells or NK cells) and was tolerable without toxicity. Methods: This is a first-in-human, open-label, dose escalation study of combination SYNCAR-001 + STK-009 in adults with relapsed or refractory (r/r) CD19+ hematologic malignancies. The objectives of this study are to evaluate the safety, preliminary efficacy, pharmacokinetics, immunogenicity, and pharmacodynamics of SYNCAR-001 + STK-009. Dose escalation follows a standard 3+3 design with STK-009 being escalated while SYNCAR-001 is held at a single fixed dose. A dose extension will enroll a cohort of patients treated at a selected dose level and indication based on dose escalation findings. SYNCAR-001 is dosed intravenously (IV) once at Day 0 and STK-009 is dosed subcutaneously (SC) over the course of the study. Participants receive a single safety lead-in dose of STK-009 prior to lymphodepletion and subsequent SYNCAR-001 infusion. After SYNCAR-001 initiation, STK-009 is dosed SC weekly for 12 weeks and then monthly for 3 months. Eligible participants include individuals with r/r chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and selected r/r B-cell non-Hodgkin lymphoma (NHL subtypes of large B cell, mantle cell, and indolent lymphoma). Prior CD19 CAR use is excluded. The primary endpoint of safety includes outcomes such as adverse events and dose-limiting toxicities. Secondary endpoints include assessments of response, pharmacokinetics, and immunogenicity. Exploratory endpoints include assessment of immune cell populations, and relevant gene/protein expression, as well as persistence, phenotype, and functionality of SYNCAR-001 in the peripheral blood and/or bone marrow in response to STK-009. Citation Format: Maria Lia Palomba, Matthew G. Mei, Paolo F. Caimi, Matthew Cortese, Marco Davila, Greg Yadnik, Nestor Huang, David Mou, Martin Oft, Paul-Joseph Aspuria, Anita Mehta-Damani, Navneet Ratti, Naiyer Rizvi, Alex Azrilevich, Ran Reshef. A Phase 1 study to evaluate the safety and tolerability of a combination autologous CD19 CAR T cell therapy (SYNCAR-001) and orthogonal IL-2 (STK-009) in subjects with relapsed or refractory  CD19 expressing hematologic malignancies (NCT05665062) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT125.