The protease MALT1 is required for chronic lymphocytic leukemia genesis in Eμ-TCL1 mice

The Bruton's Tyrosine Kinase inhibitors, such as ibrutinib, acalabrutinib, and zanubrutinib, are leading the frontline treatment in Chronic lymphocytic leukemia (CLL), but acquired resistance represents a significant clinical challenge. Our group has previously shown that targeting Mucosa-Associated Lymphoid Tissue 1 ( MALT1 ) in vitro induces apoptosis in ibrutinib-sensitive and -resistant CLL cells, suggesting the involvement of the MALT1 axis in CLL progression and survival. Here, we generated a mouse model crossbreeding the Eμ- TCL1 transgenic C57BL/6 system of spontaneous CLL with CRISPR-mediated MALT1 knockout (KO) mice to test the requirement for MALT1 in CLL development. Through genotyping, we established four cohorts with at least 25 mice per cohort: Eμ-TCL1 wt / MALT1 wt , Eμ-TCL1 wt / MALT1 +/- , Eμ-TCL1 wt / MALT1 -/- , and Eμ-TCL1 -/- / MALT1 -/- . We measured leukemia burden in these groups by measuring splenomegaly and collecting cells from the bone marrow, spleen, and peritoneal fluid following euthanasia and analyzed cell populations using flow cytometry. Our results show low/negligible CLL burden in the Eμ-TCL1 -/- / MALT1 -/- and Eμ-TCL1 wt / MALT1 -/- cohorts. Conversely, the Eμ-TCL1 wt / MALT1 wt and Eμ-TCL1 wt / MALT1 +/- groups showed higher levels of CLL cell populations in each compartment. The largest median number of cells that were positive for CLL markers were in the peritoneal fluid and were 43% and 53% respectively. There was no statistical significance between the Eμ-TCL1 wt / MALT1 wt and Eμ-TCL1 wt / MALT1 +/- cohorts in terms of CLL positive cells or levels of splenomegaly. There were significant differences between the Eμ-TCL1 -/- / MALT1 -/- and Eμ-TCL1 wt / MALT1 -/- cohorts versus the Eμ-TCL1 wt / MALT1 wt cohort in both the peritoneal fluid (p-value = 0.0162 and 0.0161 respectively) and the bone marrow (p-value = 0.0016 and 0.0017 respectively), but not the spleen. Using the Kaplan-Meier method to compare overall survival of the four groups, we showed that all cohorts survived longer than the Eμ-TCL1 wt / MALT1 wt cohort. The majority of mice in the Eμ-TCL1 -/- / MALT1 -/- and Eμ-TCL1 wt / MALT1 -/- cohorts expired due to non-CLL-related complications, namely the development of dermatological lesions and ocular ulcers. These complications led to the Eμ-TCL1wt /MALT1 +/- cohort having the largest median survival at 435 days. Therefore, we were able to show that MALT1 knockout mice experienced lower leukemia burden, as defined by positive CLL cells and splenomegaly. Our data concerning the dermatopathological complications associated with the Eμ-TCL1 -/- / MALT1 -/- and Eμ-TCL1 wt / MALT1 -/- cohorts highlights potential side effects of MALT1 inhibition and are of note due to the frequent clinical presentation of cutaneous lesions in CLL patients. Overall, our data suggest that MALT1 is required for CLL leukemogenesis and may represent a critical component in therapeutic targeting.