Preclinical Efficacy of a Novel Omacetaxine-Based Therapy for AML Expressing Somatic or Germline Mutant RUNX1

RUNX1 is the DNA-binding subunit of the core-binding factor complex and a master-regulator transcription factor (TF), which is involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 commonly occur in Myelodysplastic Syndromes (MDS), as well as in secondary (s) or de novo AML (~10%). The germline, intragenic mutations and deletions in RUNX1 cause the highly penetrant (~40%), autosomal dominant, Familial Platelet Disorder (RUNX1-FPD) by the median age of 33 years with a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function (LOF) mutations. Lack of specific targeted therapy, coupled with resistance to standard chemotherapy, may account for poorer outcome observed in AML or FPD-MM expressing somatic or germline mutant (mt) RUNX1, respectively. We previously reported that knockdown (KD) of RUNX1 (wild type and mtRUNX1) is significantly more lethal toward AML blasts progenitor cells (BPCs) expressing mt versus wild type (wt) RUNX1 or normal hematopoietic progenitor cells (HPCs), indicating greater dependency on the residual wtRUNX1. Based on higher occupancy of the BET protein BRD4 at the RUNX1 super-enhancer/enhancer (SE/E) and other SE-driven AML-relevant oncogenes, the BET inhibitor OTX015 evicted BRD4 from the chromatin and repressed RUNX1 and SE-driven oncogenes, mediating efficacy against AML BPCs expressing mtRUNX1. Utilizing the RNA-Seq signature of RUNX1-depleted (by shRNA) AML cells and conducting LINCS (Library of Integrated Network-based Cellular Signatures) 1000-CMap (Connectivity Mapping) analysis, we discovered several expression mimickers (EMs), including the top-ranking protein-translation inhibitor homoharringtonine (HHT), or its semi-synthetic analogue omacetaxine mepesuccinate (OM). We tested the pre-clinical efficacy of HHT or OM against AML cell lines (OCI-AML5 and Mono-Mac-1) and patient-derived primary AML BPCs expressing somatic mtRUNX1 or wtRUNX1. Treatment with HHT or OM (5 to 100 nM) induced significantly more lethality in OCI-AML5 and Mono-Mac-1, as well as primary AML BPCs expressing mutant versus wtRUNX1, or in normal bone marrow progenitor cells (p < 0.05). Notably, first-time ever, CRISPR-Cas9-mediated knock-in of mtRUNX1 (R174*) into wtRUNX1-expressing AML HL-60 and OCI-AML2 cells showed that, in this isogenic setting also, HHT or OM, or OTX015 (50 to 1000 nM) treatment induced significantly more lethality in mt versus wtRUNX1-expressing cells (p < 0.05). The differential lethal activity of HHT in cells was associated with marked HHT-mediated depletion of c-Myc, c-Myb, PU.1, mt/wtRUNX1 and MCL1 in mtRUNX1 expressing HL60-R174* and OCI-AML2-R174* cells. Possibly due to HHT-mediated MCL1 repression, co-treatment with HHT and venetoclax (V) (10 to 100 nM), synergistically induced in vitro apoptosis of OCI-AML5, HL60-R174* and OCI-AML2-R174* cells (CI < 1.0). Compared to vehicle control, whereas daily treatment with OM (1.0 mg/kg, SQ) alone for 3 weeks significantly reduced the leukemia burden, co-treatment with OM and V (20 mg/kg orally) exerted significantly greater reduction in AML burden due to luciferized OCI-AML2-R174* cells engrafted into NSG mice. Combined therapy with OM and V also improved survival of the mice engrafted with OCI-AML2-R174* cells, without inducing weight loss or any other toxicity. Treatment with HHT also exerted differentially greater in vitro lethality against HPCs from FPD-MM expressing germline mtRUNX1 along with other MDS/AML-associated co-mutations, compared to RUNX1-FPD HPCs. We recently also established in culture, first time ever, a germ-line mtRUNX1 (K194N)-expressing FPD/MM cell line (GMR-AML1) from the bone marrow aspirate cells of a patient with FPD-MM that also expressed mutations in BCOR, PHF6, SRFS2 and SF3B1. Exposure to HHT (10 to 100 nM) dose-dependently inhibited cell growth and induced lethality in GMR-AML1 cells. These studies highlight that treatment with OM could potentially revert hematopoiesis in FPD-MM back to the RUNX1-FPD status. Collectively, our preliminary findings strongly support the merit of testing co-treatment with OM and venetoclax in patients with AML expressing somatic mtRUNX1, as well as in FPD-MM prior to or instead of allogeneic SCT from an unrelated donor.