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.

Phase I Study of Alvocidib Followed by 7+3 (Cytarabine + Daunorubicin) in Newly Diagnosed Acute Myeloid Leukemia.

PURPOSE: Alvocidib is a cyclin-dependent kinase 9 inhibitor leading to downregulation of the antiapoptotic BCL-2 family member, MCL-1. Alvocidib has shown clinical activity in a timed sequential regimen with cytarabine and mitoxantrone in relapsed/refractory and newly diagnosed acute myeloid leukemia (AML) but has not been studied in combination with traditional 7+3 induction therapy. PATIENTS AND METHODS: A multiinstitutional phase I dose-escalation study of alvocidib on days 1-3 followed by 7+3 (cytarabine 100 mg/m2/day i.v. infusion days 5-12 and daunorubicin 60 mg/m2 i.v. days 5-7) was performed in newly diagnosed AML ≤65 years. Core-binding factor AML was excluded. RESULTS: There was no MTD on this study; the recommended phase II dose of alvocidib was 30 mg/m2 i.v. over 30 minutes followed by 60 mg/m2 i.v. infusion over 4 hours. There was one dose-limiting toxicity of cytokine release syndrome. The most common grade ≥3 nonhematologic toxicities were diarrhea (44%) and tumor lysis syndrome (34%). Overall, 69% (22/32) of patients achieved complete remission (CR). In an exploratory cohort, eight of nine (89%) patients in complete remission had no measurable residual disease, as determined by a centralized flow cytometric assay. Clinical activity was seen in patients with secondary AML, AML with myelodysplastic syndrome-related changes, and a genomic signature of secondary AML (50%, 50%, and 92% CR rates, respectively). CONCLUSIONS: Alvocidib can be safely administered prior to 7+3 induction with encouraging clinical activity. These findings warrant further investigation of alvocidib combinations in newly diagnosed AML. This study was registered at clinicaltrials.gov identifier NCT03298984.

Single-Cell Proteomic Profiling Identifies Combined AXL and JAK1 Inhibition as a Novel Therapeutic Strategy for Lung Cancer.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFß signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFß, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFß, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

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.

Combined venetoclax and alvocidib in acute myeloid leukemia.

More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25-50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models in vitro, ex vivo and in vivo. Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS.

Are Accurins the cure for Aurora kinase inhibitors?

A nanoparticle formulation of an Aurora B inhibitor increases antitumor efficacy and reduces toxicity, which may be a precedent for the use of this technology with other small molecules (Ashton et al., this issue).

Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis.

The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, calledTg(snai1b:GFP), which labels epithelial cells undergoing EMT to producesox10-positive neural crest (NC) cells. Time-lapse and lineage analysis ofTg(snai1b:GFP)embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. TreatingTg(snai1b:GFP)embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RAin vivoand raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells.

Patterns and Significance of PIM Kinases in Urothelial Carcinoma.

BACKGROUND: The Provirus integrating site Moloney murine leukemia virus (Pim) family are proteins with serine/threonine kinase activity. Studies have demonstrated overexpression of Pims in cancer. To our knowledge, only a single study has examined Pim-1 in urothelial carcinoma. The aim of this investigation was to evaluate Pim-1, Pim-2, and Pim-3 in urothelial carcinoma and assess for expression that may contribute to disease progression and serve as a site for targeted therapy. METHODS: This retrospective study included 137 cases taken from specimens from the University of Utah, Department of Pathology (2008 to 2011). Tissue was stained with antibodies against Pim-1, Pim-2, and Pim-3. Cases were classified into 3 groups, based upon current World Health Organization criteria (invasive high-grade urothelial carcinoma [IHG] [n=84], noninvasive high-grade urothelial carcinoma/carcinoma in situ [n=32], and noninvasive low-grade urothelial carcinoma [NILG] [n=21]). Cases were scored and recorded as positive or negative on the basis of the percentage of cells with cytoplasmic and/or nuclear staining. RESULTS: NILG showed higher expression of Pim-1 (relative expression rate [RER]=2.28; 95% confidence interval [CI], 0.183-0.764) and Pim-3 (RER=3.06; 95% CI, 0.423-0.816) compared with other lesions. IHG had lower expression of Pim-1 (RER=0.31; 95% CI, 0.401-0.844) and Pim-3 (RER=0.354; 95% CI, 0.322-0.816) and noninvasive high-grade urothelial carcinoma (NIHG) demonstrated increased expression of Pim-1 and (RER=2.09; 95% CI, 0.124-0.739) and Pim-2 (RER=1.70; 95% CI, 0.151-0.591). At least 1 Pim kinase protein was expressed at the following rates: 49% in IHG, 66% in NIHG, and 76% in NILG. CONCLUSION: A high percentage of urothelial carcinomas express Pim kinases. Pim expression differs in NILG, NIHG, and IHG lesions.

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.

Use of a bacteriophage lysin to identify a novel target for antimicrobial development.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

Discovery of biologically active oncologic and immunologic small molecule therapies using zebrafish: overview and example of modulation of T cell activation.

Zebrafish models continue to gain popularity as in vivo models for drug discovery. Described in this overview are advantages and challenges of zebrafish drug screening, as well as a novel in vivo screen for immunomodulatory compounds using transgenic, T cell reporting zebrafish larvae designed for discovery of compounds targeting T cell leukemia. This assay system allows rapid screening of large numbers of compounds while avoiding the pitfalls of assays based on cell cultures, which lack biologic context and are afflicted by genomic instability. The rationale for this approach is based on similarities of immature normal T cells and developmentally arrested, malignant lymphoblasts in mammalian species. The screening algorithm has been used to identify a nontoxic compound with activity in both acute leukemia models and models of multiple sclerosis, demonstrating the utility of this screening procedure.