Abstract

Hematopoietic stem/progenitor cells (HPCs) in myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in the JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, secondary AML (sAML) occurs in up to 20% of patients. Ruxolitinib (Rux), a type I, ATP-competitive, dual JAK1/2 inhibitor (JAKi), confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Because standard chemotherapy is also relatively ineffective, there is an urgent need to develop and test novel agents for the treatment of sAML. We previously reported that, although they lack additional JAK2 mutations compared to their sensitive parental counterparts, in vitro generated Rux-resistant, JAKi-persister/resistant HEL-92.1.7 and SET2 cells (JAKi-P/R) cells exhibit 3 to 10-fold lower sensitivity to Rux, and display reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TKs). We hypothesized that the relative resistance to JAKi in sAML cells would be governed by a dysregulated transcriptome and dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against JAKi-P/R sAML cells. ARV-771 recruits the E3 ligase VHL (Von Hippel-Lindau) to polyubiquitylate and mediate multiple rounds of sub-stoichiometric catalysis, leading to efficient and prolonged degradation and depletion of BETPs, including BRD4 (> 90%) in the sAML cells. Treatment with BETP-PROTAC (50 to 250 nM) markedly depleted BRD4 and BRD2, as well as caused robust attenuation of the levels of MYC, p-STAT5, Bcl-xL, PIM1 and CDK6 in JAKi-P/R sAML cells (Leukemia, 2017, PMID: 28042144). Concomitantly, BETP-PROTAC treatment mediated profound growth inhibition and apoptosis of Rux-sensitive and JAKi-P/R cultured and patient-derived CD34+ sAML cells. This translated into a significantly reduced sAML burden and improved survival, without toxicity, in the luciferase-transduced immune depleted (NSG) mice treated with ARV-771 (30 mg/kg/day for 3 weeks) versus vehicle control. In myeloid malignancies, canonical WNT-β catenin signaling is essential for survival, growth and self-renewal of leukemia stem and blast progenitor cells (BPCs). Notably, we discovered that, as compared to their sensitive controls, JAKi-P/R sAML cells exhibited higher levels of p-AKT, p-GSK3-β and increased nuclear localization of β-catenin (by confocal microscopy), associated with increased levels of β-catenin-TCF4 targets, MYC and survivin. In a previous report, we had demonstrated that treatment with the β-catenin antagonist BC-2059 that degrades and depletes nuclear levels of β-catenin and inhibits β-catenin/TCF4 transcriptional targets, resulted in apoptosis of AML stem progenitor cells (Leukemia, 2015;29:1267-78). Consistent with this, we determined that BC-2059 (50 to 100 nM) also markedly attenuated nuclear β-catenin levels, reduced expression of the β-catenin/TCF4 targets MYC and survivin, and induced in vitro apoptosis of JAKi-P/R sAML and PD CD34+ sAML cells. Co-treatment with BC-2059 and Rux synergistically induced in vitro apoptosis (combination indices < 1.0) of sAML cells, and combined therapy for 3 weeks with BC-2059 (30 mg/kg/day) and Rux (30 mg/kg/day) significantly reduced sAML burden and improved survival of NSG mice engrafted with sAML cells (p < 0.01). Recently, resistance to BETP inhibitors that evict BETPs from chromatin was shown to be at least in part due to increased activity of WNT-β-catenin/TCF4 and the increased c-Myc expression. Based on this, we also determined the in vitro and in vivo effects of co-treatment with BETP-PROTAC and BC-2059 against sAML cells. Compared to each drug alone, co-treatment with BETP-PROTAC exerted synergistic in vitro lethality against JAKi sensitive and JAKi-P/R sAML cells. These findings strongly support further in vivo development and testing of BETP-PROTAC and β-catenin antagonist-based combinations against post-MPN sAML.

Disclosures

Qian: 4Arvinas, LLC. New Haven, CT: Employment. Raina: 4Arvinas, LLC. New Haven, CT: Employment. Verstovsek: Celgene: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Lilly Oncology: Research Funding; Pfizer: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Astrazeneca: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Bristol Myers Squibb: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Incyte: Research Funding; Lilly Oncology: Research Funding; Promedior: Research Funding; Roche: Research Funding; Roche: Research Funding; Astrazeneca: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Bristol Myers Squibb: Research Funding; NS Pharma: Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.