Arthur Groy

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Antitumor Activity of GSK1904529A, a Small-molecule Inhibitor of the Insulin-like Growth Factor-I Receptor Tyrosine Kinase

Purpose: Dysregulation of the insulin-like growth factor-I receptor (IGF-IR) signaling pathway has been implicated in the development of many types of tumors, including prostate, colon, breast, pancreatic, ovarian, and sarcomas. Agents that inhibit IGF-IR activity may be useful in treatment of patients with various cancers. Experimental Design: Kinase assays were used to identify a selective small-molecule inhibitor of IGF-IR activity. The effects of this compound on IGF-IR signaling, cell proliferation, and the cell cycle were determined using a panel of cell lines. Antitumor activity was evaluated in human tumor xenografts growing in athymic mice. Inhibition of IGF-IR and the closely related insulin receptor (IR) was measured in vivo, and the effect on glucose metabolism was evaluated. Results: GSK1904529A selectively inhibits IGF-IR and IR with IC50s of 27 and 25 nmol/L, respectively. GSK1904529A blocks receptor autophosphorylation and downstream signaling, leading to cell cycle arrest. It inhibits the proliferation of cell lines derived from solid and hematologic malignancies, with multiple myeloma and Ewings sarcoma cell lines being most sensitive. Oral administration of GSK1904529A decreases the growth of human tumor xenografts in mice, consistent with a reduction of IGF-IR phosphorylation in tumors. Despite the potent inhibitory activity of GSK1904529A on IR in vitro and in vivo, minimal effects on blood glucose levels are observed in animals at doses that show significant antitumor activity. Conclusion: GSK1904529A is a promising candidate for therapeutic use in IGF-IR–dependent tumors.

A687V EZH2 Is a Driver of Histone H3 Lysine 27 (H3K27) Hypertrimethylation

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell–derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. Mol Cancer Ther; 13(12); 3062–73. ©2014 AACR.

Discovery of 4,6-bis-anilino-1H-pyrrolo[2,3-d]pyrimidines: Potent inhibitors of the IGF-1R receptor tyrosine kinase.

The evaluation of a series of 4,6-bis-anilino-1H-pyrrolo[2,3-d]pyrimidines as inhibitors of the IGF-1R (IGF-IR) receptor tyrosine kinase is reported. Examples demonstrate nanomolar potencies in in vitro enzyme and mechanistic cellular assays as well as promising in vivo pharmacokinetics in rat.

Discovery and optimization of imidazo[1,2-a]pyridine inhibitors of insulin-like growth factor-1 receptor (IGF-1R)

The optimization of imidazo[1,2-a]pyridine inhibitors as potent and selective inhibitors of IGF-1R is presented. Further optimization of oral exposure in mice is also discussed. Detailed selectivity, in vitro activity, and in vivo PK profiles of an optimized compound is also highlighted.

Abstract 3514: A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells

The isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are mutated in acute myelogenous leukemia, low-grade glioma, intrahepatic cholangiocarcinoma, and chondrosarcomas. IDH1 and IDH2 normally function to convert isocitrate into alpha-ketoglutarate. However, when these enzymes are mutated at select residues the mutant enzymes now convert α-KG into 2-hydroxyglutarate (2-HG). In normal cells, 2-HG levels are typically extremely low, but IDH1/2 mutant cells can accumulate up to 10 mM 2-HG. In an effort to counteract the neomorphic activity of mutant IDH enzymes, we identified and developed potent inhibitors of IDH1. The compounds inhibit IDH1 catalytic activity in biochemical assays and reduce 2-HG production in IDH1-mutant cell lines. Consistent with the fact that 2-HG inhibits α-KG dependent enzymes including histone demethylases and Tet family hydroxylases, these IDH1 inhibitors induce a decrease in several histone methylation marks and also DNA methylation. These data demonstrate that small molecule inhibitors can reverse many of the epigenetic effects of mutant IDH1. Note: This abstract was not presented at the meeting. Citation Format: Cynthia Rominger, Chad Quinn, Enoch Gao, Beth Pietrak, Alan Rendina, Angela Smallwood, Arthur Groy, Susan Korenchuk, Charles McHugh, Ken Wiggall, Alexander Reif, Stanley Schmidt, Hongwei Qi, Huizhen Zhao, Nestor Concha, Christopher Carpenter, Juan Luengo, Ryan Kruger, Benjamin Schwartz, Nicholas Adams, Michael T. McCabe. A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2015-3514

Abstract 961: GSK1120212 inhibits both MEK kinase activity and activation

Negative feedback loops are commonplace in signal transduction pathways, restoring homeostatic equilibrium. Inhibitory drugs to these pathways, by suppressing the negative feedback, can result in hyperactivation of upstream pathway components. MEK inhibitors have been reported to suppress ERK1/2 mediated activation of DUSPs and Sprouty, resulting in increased phosphorylation and activation of both Raf and Mek. This feedback response may negatively impact an inhibitor9s efficacy both by increasing pathway activation and by weakening the compound binding site. Here we report on studies characterizing the effect of the MEK1/2 inhibitor GSK1120212 in preventing activation of MEK by Raf kinases, a complementary effect to its inhibition of MEK kinase activity. We conducted in vitro MEK activation assays with Raf1 or BRAF and U-MEK in the presence or absence of GSK1120212, and analyzed MEK phosphorylation by ftMSMS. We observed that GSK1120212 completely prevented Raf-dependent phosphorylation of S217 on MEK1, resulting in mono-phosphorylated (S221) MEK1 (p-MEK1). We then showed that although p-MEK1(pS221) is more active than U-MEK, it is 83-fold less active than the fully-activated diphospho-MEK1 (pp-MEK1). Our inhibition study indicated that the affinity of GSK1120212 for MEK1 was reduced by S217 phosphorylation since the IC50 for pp-MEK1 was 15.3 nM, but We then assessed whether the effect of GSK1120212 on MEK1 activating phosphorylation occurs in cells. In Sk-MEL-28, A375P, and HCT116 cells, treatment with GSK1120212 results in a transient decrease in MEK phosphorylation detected by immunoblotting; however the levels increase over time for both HCT116 and A375P. Since the phospho-MEK antibodies may not distinguish the mono from diphospho MEK, we immunoprecipitated MEK1 from lysates and analyzed MEK phosphorylation by MS. As with the in vitro reaction, GSK1120212 prevented S217 phosphorylation, but not phosphorylation of S221. As suggested by immunblotting, S221 phosphorylation increased over time, presumably reflecting the block of negative feedback mechanisms. Since GSK1120212 does not directly inhibit either RAF1 or BRAF kinase activity, we believe that GSK1120212 binds to MEK in a way that specifically blocks the accessibility of S217 to Raf kinases. This is further supported by the observation that once S217 is phosphorylated, as in pp-MEK, the affinity for GSK1120212 is reduced. S217 phosphorylation likely alters the adjacent activation loop that partially defines the compound binding site. These results suggest that in Ras and Raf mutant tumors, GSK1120212 may suppress both MEK kinase activity and partially abrogate the activating effects due to negative feedback. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2011-961

Abstract C74: Reassessing IKKε as a novel oncology target

Recent years have seen increasing efforts to employ integrative genomic approaches to identify novel oncogenes. In this manner, IKBKE coding for IKKe was recently proposed as a novel oncogene, aberrantly activating the NF‐κB signaling pathway in breast cancers (Boehm et al. Cell. 2007). We pursued a series of experiments that delve more deeply into the hypothesis of IKKe as a novel oncology target; the resulting evidence challenges the proposed oncogenic mechanism. We conducted RNA interference studies on mutiple breast cancer cell lines with multiple IKBKE siRNA motifs to define a general on‐target phenotype. Consistent with published findings, we confirmed that although 4 IKBKE siRNA motifs tested significantly reduced IKKe protein expression (78–90% silencing) only 2 of these motifs significantly inhibited cell growth. We then engineered ‘non‐silenceable’ IKBKE constructs packaged in BacMam virus and were able to simultaneously silence the endogenous IKBKE and exogenously express the non‐silenceable IKKe at protein levels below, equivalent to, and significantly above the endogenous levels. We demonstrated that the antiproliferative effects of these 2 siRNAs could not be rescued by functional, exogenously expressed IKBKE, indicating likely off‐target toxicity. We further explored the correlations between IKBKE gene amplification, IKKe protein overexpression, and NF‐κB pathway activation using RNAi in conjunction with an NF‐κB ‐luciferase reporter assay. We observed that IKBKE amplification correlated poorly with both IKKe protein expression and NF‐κB pathway activation. Furthermore, silencing downstream components of the NF‐κB signaling pathway had little impact on the IKBKE‐amplified cells; and conversely, silencing IKBKE had no impact on NF‐κB ‐reporter activity in the IKBKE‐amplified cells. In addition, we over‐expressed the kinase‐dead IKKe (K38A), and observed no effect on tumor cell growth. Finally, using BacMam virus to serially titrate IKK , we confirmed that the level of exogenous IKKe needed to achieve a modest pathway activation (2‐fold increase in NF‐κB ‐luciferase reporter assay) was >10 times the endogenous protein level found in an IKBKE‐amplified breast cancer line. We also undertook a limited medicinal chemistry effort to arrive at several potent small molecule tool inhibitors of both IKK and TBK1 including GSK2292978A (8nM & 1nM respectively). GSK2292978A confirmably inhibited IKKe activity in a cellular mechanistic assay (IC50∼170nM). Using a 3‐day proliferation assay, we observed no evident selectivity of GSK2292978A for IKBKE‐amplified or IKKe overexpressing cell lines compared to non‐amplified cell lines. These findings resulted in our conclusion that IKKe likely does not represent a true oncogene in breast cancer and suggest the vital need for well‐designed controls in cases where RNA interference significantly defines target validation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C74.