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747 Superior Lethal Activity of Novel BET Protein Proteolysis Targeting Chimera (BETP-PROTACs) Versus Betp Bromodomain Inhibitor (BETi) Against Post-Myeloproliferative Neoplasm (MPN) Secondary (s) AML Cells

Molecular Pharmacology and Drug Resistance in Myeloid Diseases
Program: Oral and Poster Abstracts
Type: Oral
Session: 604. Molecular Pharmacology and Drug Resistance in Myeloid Diseases: Novel Epigenetic Modulators
Monday, December 5, 2016: 11:00 AM
Room 24 (San Diego Convention Center)

Dyana T Saenz, Ph.D1*, Warren Fiskus, Ph.D1, Kanak Raina, PhD2*, Taghi Manshouri, PhD3*, Kevin Coleman, PhD2*, Jim Winkler, PhD2, Yimin Qian, Ph.D2*, Andrew Crew, PhD2*, Angela Shen, MD2*, Christopher P Mill, Ph.D1*, Baohua Sun, MD, PhD1, Srdan Verstovsek, MD, PhD3, Craig Crews, Ph.D4* and Kapil N. Bhalla, MD3

1Department of Leukemia, MD Anderson Cancer Center, Houston, TX
2Arvinas, LLC, New Haven, CT
3Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
4Department of Molecular, Cellular, and Developmental Biology, Department of Chemistry, Department of Pharmacology, Yale University, New Haven, CT

Hematopoietic stem/progenitor cells (HPCs) of BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 & 2 inhibitor (JAKi), which is currently used in the therapy of MPN-MF. Treatment with R confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Prolonged exposure to R may also lead to a loss of response, causing the emergence of JAKi-resistant (JIR) sAML cells. Although they lack additional JAK2 mutations, JIR cells exhibit reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TK). Sequential genomic assessments in pre- and post-sAML transformation have revealed mutations in TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, SETBP1 and TP53 genes. Here, we determined that treatment with BETi, e.g., JQ1 or OTX015, which disrupts the binding of BRD4 with acetylated chromatin, significantly inhibits growth and induces apoptosis of JAKi-sensitive cultured, including those that express JAK2 V617F and mutant TP53, e.g., HEL92.1.7 and SET2, and patient derived (PD) CD34+ sAML cells. Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. Co-treatment with BETi and R synergistically induced apoptosis of cultured and PD CD34+ sAML cells. As compared to treatment with vehicle control, JQ1 or R treatment alone, co-treatment with JQ1 and R significantly improved the median survival of the immune-depleted mice engrafted with HEL92.1.7 cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the activity of c-MYC, NFkB and several other transcription factors. Therefore, we compared the anti-sAML activity of the novel BETP-PROTACs (BET protein-proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.), which degrade BETPs (including BRD4), with the BETi OTX015, against cultured and PD CD34+ sAML cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BETPs. At equimolar concentrations (100 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary sAML cells (p < 0.05), while sparing the CD34+ normal hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased BRD4 levels, BETP-PROTACs caused efficient and prolonged depletion of the levels of BETPs, including BRD4 (> 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. Western analyses showed that BETP-PROTAC versus BETI treatment also caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry approach showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-RB, while inducing p21 levels in the phenotypically characterized CD34+ sAML stem/progenitor cells, based on their high expression of CD90, CD244, CD123+ and TIM3Fc+. Compared to treatment with each agent alone, co-treatment with ARV-825 and R (100 to 1000 nM) was synergistically more lethal against the cultured and PD CD34+ sAML cells. We have isolated JAKi-resistant HEL92.1.7 cells (> 10-fold resistant to R; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAKi. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to BETP-PROTAC. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 is synergistically lethal against JAKi sensitive and JIR sAML cells. These findings strongly support the in vivo testing of the BETP-PROTACs alone and in combinations against post-MPN sAML. These studies are underway in our laboratory and will be presented at the ASH meetings.

Disclosures: Raina: Arvinas, LLC: Employment. Coleman: Arvinas, LLC: Employment. Winkler: Arvinas, LLC: Employment. Qian: Arvinas, LLC: Employment. Crew: Arvinas, LLC: Employment. Shen: Arvinas, LLC: Employment.

*signifies non-member of ASH