Cytokine-medated expansion of clonal hematopoiesis after CART therapy

Abstract Chimeric antigen receptor (CAR) T-cells are highly effective against relapsed and refractory B cell and plasma cell malignancies, but are associated with well-recognized acute toxicities such as cytokine release syndrome (CRS). Emerging long-term follow-up data has recently revealed that second primary malignancies (SPM) occur in CART recipients, with an incidence of 4.3% - 8.2%. Most SPM are myeloid malignancies such as myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML), representing approximately 50 - 60% of all SPM. These occur at a median of approximately 9 - 19 months after CART cell infusion. How CAR T cell therapy, which is not known to be genotoxic, influences subsequent myeloid neoplasms (SMNs) is unclear. One hypothesis suggests SMNs may arise from clones harboring mutations associated with clonal hematopoiesis (CH), whereby accumulation of somatic mutations in genes such as TET2 or DNMT3A can confer fitness advantages to individual hematopoietic stem and progenitor cells (HSPCs). Chronic exposure to inflammation can deplete healthy HSPCs, while HSPCs bearing CH mutations are thought to be protected. We postulated that severe acute inflammation, as can occur during CAR T-induced CRS, leads to selective expansion of CH HSPCs. To model the interaction of CAR T cell-mediated inflammatory factors and CH-associated mutations, we first modeled in vitro CRS by collecting supernatant from a co-culture of a CD19+ B-ALL cell line, CAR T cells targeting CD19, and autologous monocytes. This “CRS medium” contained markedly elevated concentrations of IL-1β, IL-2, IL-12p70, IL-23, TNF-α, and IFN-γ. Of these, TNFa and IFNy are frequently implicated as drivers of CH. Exposure to “CRS medium” for two weeks increased the variant allele frequency (VAF) of TET2KO ~ 2-fold and promoted the expansion of TET2KO HSPCs in culture by ~ 55-fold. VAF expansion in the presence of CRS cytokines was also observed in HSPC engineered to express IDH1 R132H and DNMT3A R749C (2x and 1.5x increases observed respectively). We then obtained bone marrow mononuclear cells (BMMC) harboring the CH-associated DNMT3AR882H mutation from a patient with MDS . These BMMC were incubated in serum obtained from patients who received CART19 for lymphoma before (“baseline serum”) or during (“CRS serum”) clinically-defined episodes of CRS. After 48 hours in patient serum, BMMC were plated in Methocult media for a 14 day colony assay. Cells exposed to CRS serum for 48 hours prior to plating exhibited ~2x increase in DNMT3A mutation frequency, while cells incubated with pre-CRS serum exhibited a slight decrease. Additionally, patient-derived TET2 mutant BMMC exhibited variable mutation dynamics, with two out of three samples showing mutation expansion after incubation in CRS serum. To understand the signals driving these increases in colony count and mutation frequency, we performed single cell RNA sequencing on DNMT3A mutant MDS BMMC exposed to baseline or CRS serum for 48 hours. This analysis revealed that CRS serum drives significant increases in transcription of cytokine response pathways, particularly type 1 interferon and TNF response pathways. These findings suggest that inflammatory cytokines produced by CAR T cells influence the survival and proliferation of CH mutation-bearing cells. Our results may provide an explanation for increasing reports of SMNs in patients receiving CAR T cell therapy, and highlight specific pathways that may drive this adverse outcome.