Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia.

The importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

Quantifying requirements for mitochondrial apoptosis in CAR T killing of cancer cells.

Chimeric antigen receptor (CAR) T cell therapy is an FDA-approved treatment for several hematologic malignancies, yet not all patients respond to this treatment. While some resistance mechanisms have been identified, cell death pathways in target cancer cells remain underexplored. Impairing mitochondrial apoptosis via knockout of Bak and Bax, forced Bcl-2 and Bcl-XL expression, or caspase inhibition protected several tumor models from CAR T killing. However, impairing mitochondrial apoptosis in two liquid tumor cell lines did not protect target cells from CAR T killing. We found that whether a cell was Type I or Type II in response to death ligands explained the divergence of these results, so that mitochondrial apoptosis was dispensable for CART killing of cells that were Type I but not Type II. This suggests that the apoptotic signaling induced by CAR T cells bears important similarities to that induced by drugs. Combinations of drug and CAR T therapies will therefore require tailoring to the specific cell death pathways activated by CAR T cells in different types of cancer cells.