David Hollenback

Lysophosphatidic acid acyltransferase-β: a novel target for induction of tumour cell apoptosis

Phosphatidic acid (PA) is a component of cellular membranes that is also a mediator of certain cell signalling functions associated with oncogenesis. These include ras/raf/Erk and Akt/mTor [1-3]. The authors have investigated whether it would be possible to interrupt these known oncogenic pathways through the inhibition of lysophosphatidic acid acyltransferase (LPAAT), an enzyme that catalyses the biosynthesis of PA. The expression and activity of the LPAAT-β isoform are elevated in human tumours, and the respective gene displays transforming capacity when overexpressed in vitro. Inhibition by either genetic means or by isoform-specific small molecules results in a block to cell signalling pathways and apoptosis. Furthermore, the small-molecule inhibitors of LPAAT-β are not cytotoxic to a number of normal cell types, including primary bone marrow progenitors, indicating a differential dependence of tumour cells on LPAAT-β function. These discoveries indicate that LPAAT-β represents a potential novel cancer therapy target.

Induction of Apoptosis Using Inhibitors of Lysophosphatidic Acid Acyltransferase-β and Anti-CD20 Monoclonal Antibodies for Treatment of Human Non-Hodgkin's Lymphomas

Purpose: Lysophosphatidic acid acyltransferase-β (LPAAT-β) is a transmembrane enzyme critical for the biosynthesis of phosphoglycerides whose product, phosphatidic acid, plays a key role in raf and AKT/mTor-mediated signal transduction. Experimental Design: LPAAT-β may be a novel target for anticancer therapy, and, thus, we examined the effects of a series of inhibitors of LPAAT-β on multiple human non–Hodgkins lymphoma cell lines in vitro and in vivo. Results: We showed that five LPAAT-β inhibitors at doses of 500 nmol/L routinely inhibited growth in a panel of human lymphoma cell lines in vitro by >90%, as measured by [3H]thymidine incorporation. Apoptotic effects of the LPAAT-β inhibitors were evaluated either alone or in combination with the anti-CD20 antibody, Rituximab. The LPAAT-β inhibitors induced caspase-mediated apoptosis at 50 to 100 nmol/L in up to 90% of non–Hodgkins lymphoma cells. The combination of Rituximab and an LPAAT-β inhibitor resulted in a 2-fold increase in apoptosis compared with either agent alone. To assess the combination of Rituximab and a LPAAT-β inhibitor in vivo, groups of athymic mice bearing s.c. human Ramos lymphoma xenografts were treated with the LPAAT-β inhibitor CT-32228 i.p. (75 mg/kg) daily for 5 d/wk × 4 weeks (total 20 doses), Rituximab i.p. (10 mg/kg) weekly × 4 weeks (4 doses total), or CT-32228 plus Rituximab combined. Treatment with either CT-32228 or Rituximab alone showed an approximate 50% xenograft growth delay; however, complete responses were only observed when the two agents were delivered together. Conclusions: These data suggest that Rituximab, combined with a LPAAT-β inhibitor, may provide enhanced therapeutic effects through apoptotic mechanisms.

Antileukemic Activity of Lysophosphatidic Acid Acyltransferase-β Inhibitor CT32228 in Chronic Myelogenous Leukemia Sensitive and Resistant to Imatinib

Purpose: Lysophosphatidic acid acyltransferase (LPAAT)-β catalyzes the conversion of lysophosphatidic acid to phosphatidic acid, an essential component of several signaling pathways, including the Ras/mitogen-activated protein kinase pathway. Inhibition of LPAAT-β induces growth arrest and apoptosis in cancer cell lines, implicating LPAAT-β as a potential drug target in neoplasia. Experimental Design: In this study, we investigated the effects of CT32228, a specific LPAAT-β inhibitor, on BCR-ABL-transformed cell lines and primary cells from patients with chronic myelogenous leukemia. Results: CT32228 had antiproliferative activity against BCR-ABL-positive cell lines in the nanomolar dose range, evidenced by cell cycle arrest in G2-M and induction of apoptosis. Treatment of K562 cells with CT32228 led to inhibition of extracellular signal-regulated kinase 1/2 phosphorylation, consistent with inhibition of mitogen-activated protein kinase signaling. Importantly, CT32228 was highly active in cell lines resistant to the Bcr-Abl kinase inhibitor imatinib. Combination of CT32228 with imatinib produced additive inhibition of proliferation in cell lines with residual sensitivity toward imatinib. In short-term cultures in the absence of growth factors, CT32228 preferentially inhibited the growth of granulocyte-macrophage colony-forming units from chronic myelogenous leukemia patients compared with healthy controls. Conclusion: These data establish LPAAT-β as a potential drug target for the treatment of BCR-ABL-positive leukemias.

Preclinical Evaluation of AMG 925, a FLT3/CDK4 Dual Kinase Inhibitor for Treating Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D–dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4–RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses. Mol Cancer Ther; 13(4); 880–9. ©2014 AACR.

Abstract 2861: Validation of PERK as an oncology target: A role for the unfolded protein response in cancer

The Unfolded Protein Response (UPR) is a cellular stress response to stressors that induce accumulation of unfolded proteins in the endoplasmic reticulum (aka ER stress). The UPR protects cells from ER stress by increasing the capacity of the ER and attenuating bulk translation. Intense or unresolved ER stress induces apoptosis through pro-apoptotic factors like CHoP. The UPR is activated in tumors, especially those of hematological origin. PERK, a UPR sensor-kinase, is highly active in these settings and might be an attractive target in oncology. We have generated multiple potent, selective PERK inhibitor scaffolds. Low doses of PERK inhibitor ( Emerging data might provide a solution to these challenges. PERK IP-kinase assays demonstrate that compound binding at any dose activates PERK and this activity is retained after compound removal. Exposure modeling in vitro demonstrates that transient dosing followed by compound removal results in a conventional sigmoidal dose-response curve for viability. Intermittent dosing in vivo results in CHoP induction and tumor growth inhibition even at very high doses of PERK, consistent with PERK activation following compound clearance. These findings suggest that optimized scheduling might drive robust tumor growth inhibition with reduced risk of toxicity and facilitate a standard clinical dose escalation. Citation Format: Ken Dellamaggiore, Petia Mitchell, Ji-Rong Sun, Jeffrey Jones, Tony Muchamuel, David Hollenback, Seifu Tadesse, Shon Booker, Fang-Tsao Hong, Adrian Smith, Mark Rose, Pedro Beltran, James R. Lipford. Validation of PERK as an oncology target: A role for the unfolded protein response in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2861.

Abstract A279: Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor.

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard care treatment, the disease eventually relapses. Activating FLT3 mutations are found in approximately 30% of AML patients. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here preclinical evaluation of AMG 925, a potent, selective and bioavailable FLT3/CDK4 dual kinase inhibitor. The compound inhibited AML xenograft tumor growth by >99% without detectable body weight loss. AMG 925 was also found to inhibit FLT3 mutants (e.g, D835Y) that are resistant to the current FLT3 inhibitors (e.g., quizartinib/AC220, sorafenib). CDK4 is a cyclinD-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-Rb pathway in cancer development has been well established. CDK4 specific inhibitors are being developed for treating Rb positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve clinical response rates. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A279. Citation Format: Kang Dai, Kathleen Keegan, Zhihong Li, Ma Ji, Cong Li, John Eksterowicz, Coberly Suzanne, David Hollenback, Margret Weidner, Justin Huard, Lingming Liang, Grace Alba, Jessica Orf, Mei-Chu Lo, Sharon Zhao, Rachel Ngo, Ada Chen, Lily Liu, Timothy Carlson, Lawrence R. McGee, Julio Medina, Alexander Kamb, Dineli Wickramasinghe. Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A279.

Abstract 2351: CDK4/FLT3 dual inhibitors as potential therapeutics for acute myeloid leukemia.

CDK4 is a cyclin D dependent kinase that promotes cell cycle progression in a broad range of tumor types by phosphorylating the tumor suppressor retinoblastoma protein (Rb) and releasing transcription factor E2F. Critical involvement of the cyclin D-CDK4-Rb pathway in carcinogenesis is strongly supported by a large amount of genetic evidence. In addition, promoter methylation with consequent silencing of expression of the CDK4 inhibitor, p15, has been reported in 44-60% of acute myeloid leukemia (AML) patients. It is also well established that constitutive activation of the tyrosine kinase FLT3 via mutation contributes to the development of AML, with 30% of AML carrying such activating mutations. FLT3 tyrosine kinase inhibitors used as single agents reduce peripheral blood and bone marrow blasts in only a minority of AML patients, and the effect tends to be transient. This may be due to insufficient FLT3 inhibition, the selection of drug-resistant clones, or the independence of the cell on FLT3 signaling for proliferation and survival. In preclinical models, a synergistic effect of CDK4 inhibition and FLT3 inhibition resulting in increased apoptosis of AML cell lines was reported (Wang et al., Blood, 2007). From a HTS hit through SAR optimization led to AM-5992, a potent and orally bioavailable dual inhibitor of CDK4 and FLT3 including all FLT3 mutants reported to date. AM-5992 inhibits the proliferation of a panel of human tumor cell lines including MDA-MB-435(Rb+), colo-205(Rb+), U937(FLT3WT) and induced cell death in MOLM13(FLT3ITD), MV4-11(FLT3ITD), and even in MOLM13(FLT3ITD, D835Y) which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. In mouse models of leukemia using cells with the FLT3ITD mutation, AM-5992 treatment at 150 mpk qd on days 6-16 after leukemia cell injection significantly reduced the leukemia burden and prolonged survival 11 days over that of vehicle controls. Collectively, these data support the hypothesis that simultaneously inhibition of CDK4 and FLT3 may improve the durability of clinical response in AML; and consequently that this hypothesis should be tested in the clinic. Citation Format: Zhihong Li, Kang Dai, Kathleen Keegan, Ji Ma, Mark Ragains, Jacob Kaizerman, Dustin McMinn, Jiasheng Fu, Benjamin Fisher, Michael Gribble, Lawrence R. McGee, John Eksterowicz, Cong Li, Lingming Liang, Margaret Weidner, Justin Huard, Robert Cho, Timothy Carlson, Grace M. Alba, David Hollenback, John Hill, Darrin Beaupre, Alexander Kamb, Dineli Wickramasinghe, Julio C. Medina. CDK4/FLT3 dual inhibitors as potential therapeutics for acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2013-2351