Modelling acquired resistance to DOT1L inhibition exhibits the adaptive potential of KMT2A-rearranged Acute Lymphoblastic Leukemia' [RNA-seq]
KMT2A-rearranged acute lymphoblastic leukemia (ALL) is an aggressive type of leukemia which represents the most common form diagnosed in infancy. Oncogenic KMT2A-fusion proteins recruit histone methyltransferase DOT1L which leads to misplaced H3K79 methylation inducing an abnormal transcriptomic landscape that favors leukemia development. Hence, inhibition of DOT1L represents an attractive therapeutic strategy. Unfortunately, the first-in-class DOT1L inhibitor pinometostat resulted in the development of resistance. To understand this resistance we established acquired resistance to DOT1L inhibition in a pediatric KMT2A::AFF1+ B-ALL cell line model that stably became ~35-fold more resistant to pinometostat. Interestingly, while becoming almost completely independent of DOT1L-mediated H3K79 methylation, these cells remained fully dependent on the physical presence of DOT1L, HOXA9, as well as the KMT2A::AFF1 fusion protein. Further characterization of these cells using RNA-, ChIP-, and ATAC-sequencing analyses revealed that acquired resistance to DOT1L inhibition leads to a selective loss of KMT2A-fusion driven epigenetic regulation and expression of genes such as PROM1 (encoding the hematopoietic/leukemia stem cell marker CD133) and its enhancer TAPT1, as well as other putative KMT2A::AFF1 target genes such as RUNX2, PRSS12, ZC3H12, and GNAQ. In contrast, the levels of H3K79me2 and expression of other KMT2A::AFF1 target genes, including HOXA9, MEIS1, and CDK6 remained unaffected. Concomitantly, pinometostat-resistant KMT2A::AFF1+ B-ALL cells showed upregulation of genes associated with a myeloid immunophenotype, including CD33, LILRB4/CD85k, MPEG1, CCL5, and LIMK1.  Taken together, we here present a valuable model to study the adaptive potential of KMT2A-rearranged ALL upon losing dependency on one of its main oncogenic properties. Overall design: Acquired resistance to DOT1L inhibitor pinometostat was induced in the KMT2A::AFF1+ ALL cell line SEM, resulting in the pinometostat-resistant daughter line SEMPINO_RES. To characterize our SEMPINO_RES cells and gain insights into the underlying mechanisms of acquired resistance to DOT1L inhibition, we performed RNA-sequencing (RNA-seq), ChIP-sequencing for KMT2A, AFF1, H3K4me3, H3K79me2, and H3K27ac, as well as ATAC-sequencing on SEM and SEMPINO_RES cells cultured in both the absence and presence of 50 µM pinometostat for 7 days.