High Fatality CBFA2T3-GLIS2 Pediatric Acute Megakaryoblastic Leukemia Is Sensitive to BCL-XL Inhibition

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of pediatric acute myeloid leukemia (AML) with dismal survival prognosis in children below 3 years of age (<40% of cure rates). In two thirds of cases, recurrent and mutually exclusive oncogenic fusions are detected, such as CBFA2T3-GLIS2 (CG2) or NUP98 rearrangements (e.g. NUP98-KDM5A), which are considered the transformative event in this disease. The development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material, which is among other things attributed to myelofibrosis and the rarity of primary patients. Therefore, our research group has engineered synthetic human models of high-fatality pediatric AMKL with different oncogenic fusions that faithfully phenocopy the disease in a patho-physiological context. In recent years, inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, entered the therapeutic arena in adult AML and pediatric leukemia with promising results. The molecules emphasized on are Venetoclax (a specific inhibitor of BCL2) and Navitoclax (broader affinity to BCL2, BCL-XL and BCL-W). Even though we see promising results in other leukemia, these agents were so far not investigated as potential therapies in pediatric high-fatality AMKL. To first overcome the paucity of patient material for research, we generated synthetic models of high-risk AMKL by transducing cord-blood derived hematopoietic stem and progenitor cells (HSPC) with lentiviral particles carrying the CBFA2T3-GLIS2 oncogenic fusion, followed by xenotransplantation into NSG mice. This approach generated leukemia with a latency between 9.9 and 36.1 weeks in primary mice which are able to sustain serial rounds of transplantation in recipient mice (up to 5 rounds tested). Furthermore, leukemic blasts immunophenotypically recapitulate AMKL, demonstrating expression of megakaryopoietic markers such as CD41, CD61 and CD56 that are even maintained after in vitro culture of 6 days and subsequent transplantation. Furthermore, we derived a CG2 specific gene expression signature from transcriptomic profiling of our models as well as patient samples that showed high expression of NCAM1, BMP2, ERG and low GATA1 levels, as reported for CG2 leukemia. Remarkedly, one of the specific up regulated genes in our signature for CG2 was pro-survival factor BCL2. In addition, our models are located in the megakaryocytic-erythroid differentiation space, as assessed by single cell RNAseq, and similar to their healthy counterparts of the megakaryopoietic lineage they demonstrated high expression of BCL-XL on RNA and protein level, in contrast to AML samples. Based on the aberrant expression of BCL2 in our synthetic leukemia models and lineage related expression of BCL-XL, we set out to test Venetoclax and Navitoclax in the context of AMKL. We identified Navitoclax (inhibitor of BCL2, BCL-XL and BCL-W) as a therapeutic vulnerability in our models of AMKL (CG2 and NUP98 rearranged), whereas our cells demonstrated resistance to treatment with Venetoclax. We further investigated the molecular mechanism and showed that shRNA mediated knock-down or inhibition of BCL-XL, by either Navitoclax or the BCL-XL specific proteasomal degrader DT2216, results in significant induction of apoptosis in our models of AMKL. On the contrary, genetic or pharmacological inhibition of BCL2 did not induce apoptosis in our synthetic models of AMKL, even at doses as high as 10µM. Furthermore, a genotype matched leukemic model of NUP98-KDM5A, which was presenting as monocytic (CD68+LYZ+) AML instead of AMKL, was resistant to either treatment with Venetoclax or Navitoclax whereas ML2, a MLL rearranged AML cell line, showed increased sensitivity to both Venetoclax and Navitoclax treatment. Moreover, in vivo treatment of a xenografted CG2 AMKL model with Navitoclax reduced leukemic burden in mice in bone-marrow and spleen in comparison to vehicle treated controls. In conclusion, we generated a model of rare CG2 pediatric high-fatality leukemia that faithfully mimics the patient situation and allows biomass generation for large scale multi-omic approaches. Furthermore, we demonstrate a lineage- and genotype specific targetable dependency of pediatric AMKL towards inhibition of BCL-XL but not BCL2, that could be transferred to the clinic as a novel therapeutic option in high-risk infant leukemia.

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