Dissecting and targeting a novel immunometabolic checkpoint in multiple myeloma

Disease progression in multiple myeloma (MM) depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth. For example, in bone marrow microenvironment there is arginine shortage, as consequence of myeloid-derived suppressor cells (MDSC) expansion, and T-cells miss co-stimulatory signaling, engage PD-1 to bind PDL1 expressed on MDSC and neoplastic cells becoming anergic and dysfunctional. Based on our strong data generated in vitro, we hypothesize that upon arginine deprivation, PD1 and PDL1 are induced in MM, to evade immune response, through engagement of PDL1 to PD1+exhausted T cells, and to aggregate other MM cells, conferring the typical focal involvement of the bone marrow. This is part of MM adaptive response to aminoacid deprivation that involves the specific GCN2 circuitry, part of integrated stress response, that if dissected can provide novel means of nononcogene synthetic lethality.

Hypothesis

We hypothesize that MDSC induce an arginine and tryptophan shortage in the microenvironment. This amino acids deprivation induces anergy of T lymphocytes on one side while on the other side it elicits an adaptive response of MM cells by triggering the GCN2 downstream signaling, to promote autophagy. When further amino-acid shortage occurs, sensitivity to proteasome inhibition relies on the magnitude of the elicited adaptive response. Dissecting and potentially targeting the GCN2/ATF4 downstream signaling can reveal new synthetic lethality mechanisms to improve long-term outcome in MM.

Aims

Integrating in-vitro and in-vivo models we propose to: dissect the molecular basis that associates GCN2 activation to IRF4 availability and PD1 over-expression in MM cells, upon amino-acid deprivation; reveal if targeting arginine and tryptophan shortage in the environment could improve outcome in MM.

Experimental Design

We will use four pre-clinical models: 1.Three MM cells lines (MM1.S, U266, OPM2), chosen on the basis of cytogenetics, bortezomib sensitivity, IRF-4 and GCN2 basal expression, will be cultured in acute and chronic arginine or tryptophan shortage to evaluate at different time points (1-12, 24-48-72-96 hours). Genetic manipulation using CRISPRi/a-Cas9 technology will be used to modify or disrupt gene dosage of GCN2, ATF4, CHOP, IRF4 and PD1. 2.time and size of the engraftment in a model based on immunocompetent adult zebrafish challenged with human myeloma cell lines intra-peritoneal injected in presence or not of Arg-1/IDO1 inhibitors, associated or not to bortezomib 3.time and size of the engraftment, synergism of bortezomib, Arg-1/IDO1 and GCN2 inhibitors in two murine models to confirm findings.

Expected Results

Upon arginine deprivation, PD1 and PDL1 should be induced in MM, to evade immune response, through engagement of PDL1 to PD1+exhausted T cells, and to aggregate other MM cells, conferring the typical focal involvement of the bone marrow. Adaptive response to arginine shortage should be targeted by GCN2 inhibitors combined to Arg-1 inhibitor and bortezomib for long-term control of the disease.

Impact on Cancer

Restoring immune system function in MM is an unmet clinical need. Our project will investigate a new molecular circuitry to enhance long-term control of the disease.