Temporally Controlled MYC Overexpression in Chronic Lymphocytic Leukemia Accelerates Progression and Promotes Large B Cell Lymphoma Transformation

Background: Richter's transformation (RT) is the progression of chronic lymphocytic leukemia (CLL) to a high-grade lymphoma, usually resembling diffuse large B-cell lymphoma, with a poor prognosis of 6-12 months. The molecular mechanisms behind CLL-to-RT are unclear but often involve MYC hyperactivation through alterations, such as t(8;14), del(MGA), MYC/n-MYC gain, and mutations or overexpression of MYC regulators (e.g., NOTCH1, PRMT5). Previous efforts to model MYC overexpression in CLL (Eμ- TCL1xMyc) led to concurrent development of CLL and lymphoma rather than clonal transformation, possibly due to constitutive overexpression oncogenic drivers. Thus, we aimed to evaluate the oncogenic and immunologic effects of inducible B cell-specific overexpression of human MYC at various CLL development stages.

Methods: We created the triple transgenic iMYC-TCL1 murine model of temporally controlled MYC overexpression in B cells of the Eμ-TCL1 CLL model by crossing it to the R26StopFLMYC and CD19.cre-ERT2 murine models (R26StopFLMYC^het. CD19.cre-ERT2^het. Eμ-TCL1^hemi). The R26StopFLMYC allows for the conditional overexpression of both MYC and an inactive human CD2 marker. Cre recombination was induced by administration of Tamoxifen (TAM, 110mg/kg) or vehicle (Veh, corn oil) via oral gavage (QD x 5 days). Mice were monitored monthly by flow cytometry for circulating CLL cells (cCLL; Cd19+Cd5+) and enrolled into disease load groups (<5% and >20% of cCLL) and treated with TAM or Veh. Limiting cell RNAseq (lcRNAseq) was performed on circulating malignant B cell populations (Cd19+Cd5+, Cd19+Cd5+hCD2+, and Cd19+hCD2+) for evaluation of BCR gene expression and global DEGs.

Results: Induction of MYC in pre-leukemic mice (<5% cCLL) (n=9 TAM, n=6 Veh) resulted in rapid disease expansion with 5/9 TAM vs 0/6 Veh mice achieving >25% circulating disease after 3.2 months (p<0.03). The MYC induced population had increased cell size (FSC) by flow cytometry. Since the clonal relationship of RT to CLL is a key prognostic factor, we assessed MYC induction's clonal dynamics by analyzing malignant B cell populations in TAM-treated mice using FACS and lcRNAseq (n=4). Clonal relationship between Cd19+Cd5+ and Cd19+Cd5+hCD2+ was established in all TAM treated mice with light chain CDR3 sequences of the dominant Cd19+Cd5+hCD2+ clone emerging from a pre-existing Cd19+Cd5+ clone. Furthermore, the Cd19+Cd5+hCD2+ population shows enrichment of E2F target genes (NES: 1.29, p < 0.05) and decreased interferon gamma response genes (NES: -1.56, p < 0.05) when compared to CLL-like B cells (Cd19+Cd5+) as has been previously shown in RT vs CLL tumors. Intestinal tumors composed of Cd19+Cd5+ and Cd19+Cd5-hCD2+ cells were seen in 4/9 TAM treated mice and was a phenotype seen only within this group.

Next, to mimic RT development with MYC hyperactivation post-CLL development we induced the iMYC-TCL1 model at >20% cCLL (n=14, 7 per treatment group). Induction led to a rapid expansion of Cd19+Cd5+hCD2+ cells infiltrating the spleen, lymph nodes, and marrow of mice, as early as 4 weeks post-TAM. Splenic B cells in TAM treated mice at ERC were predominantly Cd19+Cd5+hCD2+ (mean: 72% vs 5% Cd19+Cd5+, p<0.01). MYC induced, Cd19+Cd5+hCD2+, cells show increased Pdl1 expression relative to Cd19+Cd5+hCD2- cells. Significant median survival differences were observed (TAM: 80 vs Veh: 98 days post-induction, p<0.05). Histopathologic analyses are currently ongoing.

To assess the impact of MYC induction on the tumor microenvironment, we analyzed circulating T cell profiles in post-leukemic TAM and Veh treated mice. Veh treated mice had predominantly memory CD8+ T cells, while TAM treated mice showed reduced T cell abundance but increased CD8+ T cell diversity (memory, effector, naïve).

Conclusion: Induction prior to CLL development leads to the development and expansion of large B cells, clonally related to the CLL B cell population, and akin to RT, upregulate E2F target genes. Notably, GI tumors were observed only in the preleukemic TAM-treated mice which demonstrates the impact of disease burden prior to induction in iMYC-TCL1 mice. MYC induction post-CLL development further accelerates clonal disease expansion, upregulates Pdl1, skews the T cell repertoire, and shortens overall survival. B cell-specific overexpression of MYC in the iMYC-TCL1 model imparts features of RT and serves as a RT disease model and therapeutic development platform.

Woyach:Pharmacyclics: Consultancy, Research Funding; Newave: Consultancy; Merck: Consultancy; Loxo Lilly: Consultancy; Janssen: Research Funding; Genentech, Inc.: Consultancy; BeiGene: Consultancy; AstraZeneca: Consultancy; AbbVie: Research Funding; Schrodinger: Research Funding; Morphosys: Research Funding.