Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells
Authors: Fiskus Warren , Sharma Sunil , Abhyankar Sunil , McGuirk Joseph , Bearss David J. , Bhalla Kapil 
Published: October 2019

Abstract 868

LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML.

Disclosures:

Sharma: Salarius Pharmaceuticals: Equity Ownership.