TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Genetic ablation of Pim1 or pharmacologic inhibition with TP-3654 ameliorates myelofibrosis in murine models.

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasm (MPN). The JAK inhibitor Ruxolitinib can reduce constitutional symptoms but it does not substantially improve bone marrow fibrosis. Pim1 expression is significantly elevated in MPN/MF hematopoietic progenitors. Here, we show that genetic ablation of Pim1 blocked the development of myelofibrosis induced by Jak2V617F and MPLW515L. Pharmacologic inhibition of Pim1 with a second-generation Pim kinase inhibitor TP-3654 significantly reduced leukocytosis and splenomegaly, and attenuated bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of MF. Combined treatment of TP-3654 and Ruxolitinib resulted in greater reduction of spleen size, normalization of blood leukocyte counts and abrogation of bone marrow fibrosis in murine models of MF. TP-3654 treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, we show that TP-3654 treatment significantly inhibits mTORC1, MYC and TGF-ß signaling in Jak2V617F mutant hematopoietic cells and diminishes the expression of fibrotic markers in the bone marrow. Collectively, our results suggest that Pim1 plays an important role in the pathogenesis of MF, and inhibition of Pim1 with TP-3654 might be useful for treatment of MF.

AXL Inhibitor TP-0903 Reduces Metastasis and Therapy Resistance in Pancreatic Cancer.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Chronic lymphocytic leukemia cells from ibrutinib treated patients are sensitive to Axl receptor tyrosine kinase inhibitor therapy.

Earlier we have shown the expression of a constitutively active receptor tyrosine kinase Axl in CLL B-cells from previously untreated CLL patients, and that Axl inhibitor TP-0903 induces robust leukemic B-cell death. To explore whether Axl is an effective target in relapsed/refractory CLL patients, we analyzed CLL B-cells obtained from CLL patients on ibrutinib therapy. Ibrutinib-exposed CLL B-cells were treated with increasing doses (0.01- 0.50µM) of a new formulation of high-affinity Axl inhibitor, TP-0903 (tartrate salt), for 24 hours and LD50 doses were determined. Sensitivity of CLL B-cells was compared with known prognostic factors and effect of TP-0903 was also evaluated on Axl signaling pathway in CLL B-cells from this cohort. We detected sustained overexpression of Axl in CLL B-cells from CLL patients on ibrutinib treatment, suggests targeting Axl could be a promising strategy to overcome drug resistance and killing of CLL B-cells in these patients. We found that CLL B-cells from sixty-nine percent of relapsed CLL patients actively on ibrutinib therapy were found to be highly sensitive to TP-0903 with induction of apoptosis at nanomolar doses (≤0.50 µM). TP-0903 treatment effectively inhibited Axl phosphorylation and reduced expression levels of anti-apoptotic proteins (Mcl-1, XIAP) in ibrutinib exposed CLL B-cells. In total, our in vitro preclinical studies showing that TP-0903 is very effective at inducing apoptosis in CLL B-cells obtained from ibrutinib-exposed patients supports further testing of this drug in relapsed/refractory CLL.

Fragment-based design, synthesis, biological evaluation, and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors.

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.

Targeted Axl Inhibition Primes Chronic Lymphocytic Leukemia B Cells to Apoptosis and Shows Synergistic/Additive Effects in Combination with BTK Inhibitors.

PURPOSE: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL. EXPERIMENTAL DESIGN: Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination. RESULTS: CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903-induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis. CONCLUSIONS: Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL.

Inhibition of Nek2 by small molecules affects proteasome activity.

BACKGROUND: Nek2 is a serine/threonine kinase localized to the centrosome. It promotes cell cycle progression from G2 to M by inducing centrosome separation. Recent studies have shown that high Nek2 expression is correlated with drug resistance in multiple myeloma patients. MATERIALS AND METHODS: To investigate the role of Nek2 in bortezomib resistance, we ectopically overexpressed Nek2 in several cancer cell lines, including multiple myeloma lines. Small-molecule inhibitors of Nek2 were discovered using an in-house library of compounds. We tested the inhibitors on proteasome and cell cycle activity in several cell lines. RESULTS: Proteasome activity was elevated in Nek2-overexpressing cell lines. The Nek2 inhibitors inhibited proteasome activity in these cancer cell lines. Treatment with these inhibitors resulted in inhibition of proteasome-mediated degradation of several cell cycle regulators in HeLa cells, leaving them arrested in G2/M. Combining these Nek2 inhibitors with bortezomib increased the efficacy of bortezomib in decreasing proteasome activity in vitro. Treatment with these novel Nek2 inhibitors successfully mitigated drug resistance in bortezomib-resistant multiple myeloma. CONCLUSION: Nek2 plays a central role in proteasome-mediated cell cycle regulation and in conferring resistance to bortezomib in cancer cells. Taken together, our results introduce Nek2 as a therapeutic target in bortezomib-resistant multiple myeloma.

Activators of PKM2 in cancer metabolism.

Pyruvate kinase converts phosphoenolpyruvate to pyruvate, catalyzing the rate-limiting step of glycolysis. The M1 isoenzyme of pyruvate kinase (PKM1) is found in adult tissues; whereas, PKM2 is a splicesome variant found in embryonic and cancer cells. PKM2 expression in malignant cells is a result of the tumor microenvironment and is responsible for maintaining a glycolytic phenotype. PKM2 has other nonmetabolic functions in malignant cells, including transcriptional coactivation and protein kinase activity. PKM2 activators have antitumor properties by inducing tetramerization of two PKM2 dimers causing PKM2 to function like PKM1. Restoring PKM2 to PKM1-like levels of activity causes reversal of the Warburg effect in cancer cells. PKM2 activators have therapeutic potential in the treatment of cancer and other metabolic diseases.

A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas.

The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

High-throughput virtual screening identifies novel N'-(1-phenylethylidene)-benzohydrazides as potent, specific, and reversible LSD1 inhibitors.

Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine methylation at residues K4 and K9 on histone H3 and is an attractive therapeutic target in multiple malignancies. Here we report a structure-based virtual screen of a compound library containing ∼2 million small molecular entities. Computational docking and scoring followed by biochemical screening led to the identification of a novel N'-(1-phenylethylidene)-benzohydrazide series of LSD1 inhibitors with hits showing biochemical IC50s in the 200-400 nM range. Hit-to-lead optimization and structure-activity relationship studies aided in the discovery of compound 12, with a Ki of 31 nM. Compound 12 is reversible and specific for LSD1 as compared to the monoamine oxidases shows minimal inhibition of CYPs and hERG and inhibits proliferation and survival in several cancer cell lines, including breast and colorectal cancer. Compound 12 may be used to probe LSD1's biological role in these cancers.

TIG1 promotes the development and progression of inflammatory breast cancer through activation of Axl kinase.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer, but the basis for its aggressive properties are not fully understood. In this study, we report that high tumoral expression of TIG1 (RARRES1), a functionally undefined membrane protein, confers shorter survival in patients with IBC. TIG1 depletion decreased IBC cell proliferation, migration, and invasion in vitro and inhibited tumor growth of IBC cells in vivo. We identified the receptor tyrosine kinase, Axl, as a TIG1-binding protein. TIG1 interaction stablilized Axl by inhibiting its proteasome-dependent degradation. TIG1-depleted IBC cells exhibited reduced Axl expression, inactivation of NF-κB, and downregulation of matrix metalloproteinase-9, indicating that TIG1 regulates invasion of IBC cells by supporting the Axl signaling pathway in IBC cells. Consistent with these results, treatment of IBC cells with the Axl inhibitor SGI-7079 decreased their malignant properties in vitro. Finally, TIG1 expression correlated positively with Axl expression in primary human IBC specimens. Our findings establish that TIG1 positively modifies the malignant properties of IBC by supporting Axl function, advancing understanding of its development and rationalizing TIG1 and Axl as promising therapeutic targets in IBC treatment.

An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance.

PURPOSE: Epithelial-mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non-small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. EXPERIMENTAL DESIGN: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. RESULTS: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. CONCLUSION: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype.

Targeting Axl and Mer kinases in cancer.

Receptor tyrosine kinases (RTK) are cell-surface transmembrane receptors that contain regulated kinase activity within their cytoplasmic domain and play an important role in signal transduction in both normal and malignant cells. The mammalian TAM RTK family includes 3 closely related members: Tyro-3, Axl, and Mer. Overexpression or ectopic expression of the TAM receptors has been detected in a wide array of human cancers. Growth arrest-specific gene 6 has been identified as the major ligand for these TAM RTKs, and its binding to the receptors has been shown to promote proliferation and survival of cancer cells in vitro. Abnormal expression and activation of Axl or Mer can provide a survival advantage for certain cancer cells. Inhibition of Axl and Mer may enhance the sensitivity of cancer cells to cytotoxic agents and would potentially be a therapeutic strategy to target cancer cells. This review elucidates the role of Axl and Mer in normal cellular function and their role in oncogenesis. In addition, we review the potential to inhibit these RTKs for the development of therapeutic targets in treatment of cancer.

In vitro and in vivo characterization of SGI-1252, a small molecule inhibitor of JAK2.

OBJECTIVE: Constitutive activation of the Janus kinase 2 (JAK2) due to a somatic mutation (JAK2(V617F)) arising in hematopoietic stem cells plays a central role in the pathophysiology of myeloproliferative neoplasms (MPNs). To investigate the hypothesis that drugs that inhibit JAK2 have therapeutic potential, we developed a small molecule inhibitor, SGI-1252, that targets the adenosine triphosphate-binding and solvent pocket of the protein. MATERIALS AND METHODS: Established cells lines each expressing different JAK2(V617F) copy numbers, a cell line transfected with wild-type and mutant JAK2, ex vivo expanded erythroid progenitor cells from patients with MPNs, and a murine xenograft model were used to characterize the activity of SGI-1252. RESULTS: In vitro studies showed that SGI-1252 potently inhibits the kinase activity of wild-type JAK2, JAK2(V617F) and JAK1, but not JAK3. SGI-1252 blocked phosphorylation of signal transducers and activators of transcription 5, a downstream target of JAK2 and inhibited expression of the JAK2-dependent antiapoptotic gene BCL-X(L). Additional studies confirmed induction of apoptosis in JAK2(V617F)-positive cell lines by SGI-1252. Moreover, cell lines transfected with either wild-type JAK2 or JAK2(V617F) were equally susceptible to the antiproliferative effects of SGI-1252 and the antiproliferative activity of SGI-1252 toward ex vivo--expanded erythroid progenitors from patients with polycythemia vera and primary myelofibrosis appeared independent of the JAK2(V617F) allele burden. Pharmacodynamic studies in a murine xenograft model demonstrated both anti-tumor activity and inhibition of signal transducers and activators of transcription 5 phosphorylation by SGI-1252, and the drug was active and well-tolerated whether delivered intraperitoneally or orally. CONCLUSIONS: Together, these studies support further development of SGI-1252 for clinical use.

Competitive enhancement of HGF-induced epithelial scattering by accessory growth factors.

HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial-mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFß, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell-cell junction disassembly and cell migration. Further, gradients of TGFß induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFß. Interestingly, VEGF suppresses TGFß-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFß might modulate HGF signaling to influence route selection during cancer progression.

Design, Synthesis and Biological Evaluation of a Series of Novel Axl Kinase Inhibitors.

The receptor tyrosine kinase AXL has emerged in recent years as an potential oncology target due to its over expression in several types of cancers coupled with its ability to promote tumor growth and metastasis. In order to identify small molecule inhibitors of AXL, we built a homology model of its catalytic domain to virtually screen and identify scaffolds displaying an affinity for AXL. Further computational and structure-based design resulted in the synthesis of a series of 2,4,5-trisubstitued pyrimidines which demonstrated potent inhibition of AXL in vitro (IC(50) 19 nM) and strongly inhibited the growth of several pancreatic cell lines.