SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Acute Myeloid Leukemia Models

Abstract

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for ~25% of all AMLs, carries a poor prognosis, and is prone to relapse despite targeted therapy. FLT3 mutations are associated with aberrant activation of the Wnt signaling pathway, which itself is implicated in AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. These studies examined the antitumor activity of SM09419 as a single agent and in combination with targeted and standard therapies in preclinical models of FLT3-ITD AML. In MV-4-11 and MOLM-13 AML cells carrying the FLT3-ITD mutation, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt pathway-related genes (CCND1, MYC, TCF7, DVL2). The effect on cell proliferation was tested in 8 AML cell lines with varying mutation profiles as well as 26 different leukapheresis-derived primary human AML cells. Proliferation was strongly impaired by SM09419 across all tested cell lines (average EC50=0.2 + 0.048 µM]); MV-4-11 and MOLM-13 cells had EC50 of 0.049 and 0.144 µM, respectively. SM09419 also potently inhibited proliferation in all primary AML cells (average EC50=0.048 + 0.0097 µM) regardless of FLT3 mutation status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). SM09419 also induced apoptosis in MV-4-11 and MOLM-13 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with either midostaurin (FLT3 inhibitor) or venetoclax (BCL2 inhibitor) and/or azacitidine were assessed in FLT3-ITD xenograft models (n=5-6/group). In MOLM-13 xenografts, SM09419 (12.5 and 25 mg/kg) induced strong tumor growth inhibition (TGI) vs. vehicle at Day 14 (TGI 52% [p<0.05] and 74% [p<0.001], respectively). Midostaurin (50 mg/kg) induced significant TGI vs. vehicle (50%, p<0.05), which was increased when administered in combination with 12.5 mg/kg SM09419 (81%, p<0.001). In MV-4-11 xenografts, single-agent SM09419 (6.25, 12.5, and 25 mg/kg) induced significant TGI vs. vehicle (56% [p<0.05], 94%, and 95% [p<0.001], respectively) at Day 26 with tumor regression in all mice dosed at 12.5 mg/kg and 25 mg/kg. In a subsequent experiment, midostaurin (50 mg/kg) alone and combined with 6.25 mg/kg SM09419 for 23 days induced tumor regression in MV-4-11 xenografts (100% TGI vs. vehicle, p<0.0001). After treatment discontinuation, tumor regression was maintained in all mice (6/6) treated with the combination for 26 days, whereas tumor regrowth was immediately observed in midostaurin-treated mice. In another MV-4-11 xenograft study, the combination of 6.25mg/kg SM09419 with azacitidine (0.8 mg/kg QD) and/or venetoclax (25 mg/kg QD) induced significant TGI (95-98% vs. vehicle, p<0.001) with tumor regression at Day 26. Azacitidine + venetoclax induced 79% TGI (p<0.001), but no tumor regression was observed. The triple combination induced tumor regression in all mice and complete regressions in 4/6 mice (67%); it had a greater effect on slowing tumor regrowth after treatment discontinuation vs. a single agent or doublet. SM09419 alone or in combination was well tolerated in these xenograft models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation and Wnt signaling pathway activity and induced apoptosis in FLT3-ITD cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of FLT3 status. The strong in vivo antitumor effects observed as combination treatment suggest that SM09419 combined with standard therapies may provide a clinical benefit by slowing or preventing relapse in AML with a marker of poor prognosis such as FLT3-ITD. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

Structure-Guided Discovery of Potent and Selective CLK2 Inhibitors for the Treatment of Knee Osteoarthritis

“…Currently, two CLK inhibitors are in clinical trials, which suggests that nonselective CLK inhibitors may exhibit synergistic effects on disease treatment. 41 The enzyme inhibitory activity of the representative compound LQ23 (Table 3, Figure 2A) on the CLK1, CLK2, CLK3, CLK4, and DYRK1A were tested in vitro. Encouragingly, LQ23 exhibited higher inhibitory potency against CLK1/2/4 (CLK1, IC 50 = 2.1 nM; CLK2, IC 50 = 1.4 nM; CLK4, IC 50 = 3.2 nM) compared to Lorecivivint.…”

An overview of cdc2‐like kinase 1 (Clk1) inhibitors and their therapeutic indications

“…Despite the reported few differences in pocket sizes and electrostatic surface charge distributions among the active sites of Clk isoforms, 155 the development of selective inhibitors for Clk1 remains challenging due to the high sequence homology for human Clk1–Clk2, Clk1–Clk3, Clk1–Clk4 verified to be 54%, 48.4%, and 78.4%, respectively 11 . In fact, the three orally active Clk inhibitors that made progress to phase I clinical trials (structures not disclosed by authors), CTX‐712 for relapsed and refractory AML, 68 SM09419 for advanced hematologic malignancies, 69 and SM08502 for advanced solid tumors are all pan‐Clk inhibitors 65 . As can be seen from Table 17, the discovery of selective Clk1 inhibitors over Clk3 was feasible, owing to the lower degree of homology, with numerous successful examples in literature.…”

“…Clk1 inhibition has been established as a novel effective strategy in treating various types of tumors such as hypoxia-induced prostate 63 and lung cancers, 58 gastrointestinal cancer, 64,65 colorectal cancer, 66 urinary bladder carcinoma, 18,67 and several hematological malignancies. 68,69 Clk1 was shown to phosphorylate and activate splicing factor 45 (SPF45) on eight serine residues leading to stabilization of SPF45 protein levels. 20 SPF45 promotes skipping of exon 6 in Fas pre-mRNA, 70 which encodes a transmembrane domain in the Fas death receptor, resulting in the formation of a soluble Fas protein molecule (Figure 2).…”

“…The synthesized inhibitors were tested against several kinases which include CDK1, CDK2, CDK5, CK1, Clk1-4, Dyrk1A, Dyrk1B, Dyrk2, Dyrk3, and GSK3. 162 Several structural modifications were adopted to optimize the lead of this series, the unsubstituted derivative (69) showed an IC 50 of 46 nM against Clk1, (69) had good selectivity over the other tested kinases, except the Dyrk2 kinase. 162 Substitution at R 2 , R 3 , and R 5 had huge effects on either the activity or the selectivity of the pyrroloindolone; substitution at R 2 or R 5 maintained the Clk1 inhibitory potency, however, it diminished the selectivity over CDK1, CDK5, CK1, and most importantly Dyrk1A; substitution at R 3 was deleterious for Clk1 inhibitory activity.…”

“…(72) was the most potent Clk1 inhibitor of the series, however, the selectivity for Clk1 over Dyrk1A was lost, in comparison to compounds (69) and (71). 162 Kinase inhibition data for the compounds 69-72 are summarized in Table 12.…”