Phase I First-in-Human Dose Escalation Study of the oral Casein Kinase 1α and Cyclin Dependent Kinase 7/9 inhibitor BTX-A51 in advanced MDS and AML

Abstract

BTX-A51, a first-in-class oral small molecule inhibitor of casein kinase 1α (CK1α) and cyclin dependent kinase (CDK) 7 and 9, induces apoptosis of leukemic cells by activating p53 and inhibiting expression of Mcl1. Here, we report on the results of the phase 1 clinical trial of BTX-A51 in patients with relapsed or refractory AML and MDS. Thirty-one patients were enrolled into 8 dose-escalation cohorts at BTX-A51 doses ranging from 1mg to 42mg dosed three days/week for 21 or 28 days out a 28-day cycle. The recommended phase 2 dose was 21mg dosed three days/week for 4 weeks of a 28-day cycle. BTX-A51 increased expression of p53 and reduced expression of MCL1 and RNA polymerase II phosphorylation on pre- and post-treatment immunocytochemistry studies. Overall, 3 patients (10%) experienced complete remission with incomplete count recovery (CRi). All 3 responding patients had RUNX1 mutations and the CR/CRi rate for RUNX1-mutated patients receiving BTX-A51 at efficacious doses (11mg or higher) was 30%. Ex-vivo studies confirmed higher efficacy of BTX-A51 on RUNX1-mutated myeloblasts and demonstrate synergy with azacitidine and venetoclax. Although the overall efficacy was modest, this study lays the groundwork for future studies with improved patient selection and combination approaches.

Figure 1

Swimmer plot of enrolled patients by dose level and their outcomes after treatment with BTX-A51, including time to response, duration of treatment, and patient status at time of data cutoff date. RUNX1-mutated patients are indicated with a red box. CRi, Complete response with incomplete count recovery, SD, stable disease, PD, progressive disease, TS, treatment stopped, Tx, treatment failure, OT, Ongoing treatment, mg, milligrams.

Figure 2

Pharmacodynamic studies of BTX-A51. Immunostaining of MCL1, P53, RNA polymerase II phosphorylation, and histone H2AX phosphorylation on CD34+ cells isolated from pretreatment (screening) and post-treatment (Cycle 2 Day1) bone marrow specimens. Reduced expression of MCL1 and RNA polymerase II phosphorylation and increased expression of P53 and histone H2AX phosphorylation after treatment with BTX-A51. Scale bar, 10μm.

Figure 3

Bone marrow and peripheral blood blast count reduction after treatment with BTX-A51 by dose level and RUNX1 mutation status. A. Peripheral blood blast count reduction during cycle 1 among patients with detectable peripheral blood blasts at screening. B. Best bone marrow blast reduction among patients with pre and post treatment bone marrow biopsies. C. CR/CRi rates among all patients in an intention-to-treat analysis and among all RUNX1-mutated patients. RUNX-1 mutated patients are identified by color in A. and B. CR, complete remission, CRi, complete response with incomplete count recovery, mg, milligrams, ITT, Intention to treat.

Figure 4

Immunoblot analysis of 5 different human AML cell lines treated with 100nM of A51 (+) or with DMSO (−) for 24 hours. PP2Ac is a loading control. Treatment with A51 resulted in decrease in phosphorylated Ser5 and Ser2 of the C-terminal domain (CTD) of RNA polymerase II, indicating inhibition of CDK7 and CDK9 respectively. This was followed by reduced expression of common leukemia oncogenes MYC, MDM2 and MCL1 and induction of a DNA damage response and apoptosis, as evidenced by the induction of γH2AX and cleaved caspase 3. At the indicated treatment procedure, a p53 response is evident only in OCI-AML-5 (RUNX1 mutant cell line).

Figure 5

Ex vivo inhibition of primary RUNX1-mutated AML cells by BTX-A51. A. Flow sorted CD34 positive primary RUNX1-mutated AML blasts were plated onto preplated HUVEC_AD52 cells and the expansion of CD34 positive cells were calculated in the presence of BTX-A51 or Venetoclax. IC50 was calculated. B-C. NRAS/ IDH2 mutant primary AML blasts were flow sorted and cultured in preplated HUVEC_AD52 cells. The expansion of CD34 positive cells were calculated in the presence of BTX-A51. IC50 was calculated. D-E. The additive effects of BTX-A51/Venetoclax and BTX-A51/Azacytidine were illustrated (Bliss synergy score was 8.04 and 8.75, respectively). F. The synergy between BTX-A51, Venetoclax and Azacytidine was plotted. *, p≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.