Keith Mikule

Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1.

Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics.

Discovery of Potent KIFC1 Inhibitors Using a Method of Integrated High-Throughput Synthesis and Screening

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZenecas collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

Synthetic lethality of PARP inhibitors in combination with MYC blockade is independent of BRCA status in triple negative breast cancer

PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial-mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment.Significance: Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. Cancer Res; 78(3); 742-57. ©2017 AACR.

Abstract B168: The PARP inhibitor, niraparib, crosses the blood brain barrier in rodents and is efficacious in a BRCA2-mutant intracranial tumor model

Introduction The incidence of brain metastases among breast and ovarian cancer patients with germline BRCA mutations (gBRCAmut) is significantly higher than those with gBRCA wild-type tumors (gBRCAwt), and has been estimated to be greater than 50%. gBRCAmut patients with brain metastases have a worse prognosis, and limited therapeutic options. The potential of two poly-ADP-ribose polymerase 1 inhibitors, niraparib and olaparib, were evaluated for their ability to target a gBRCAmut intracranial xenograft tumor model. Methods Pharmacokinetic (pk) studies were conducted in rat to determine the brain and plasma levels of niraparib following a single oral dose of 10 or 30 mg/kg. The BRCA2-mutant Capan-1 human pancreatic cancer xenograft model was utilized to evaluate efficacy. Briefly, randomized cohorts of Balb/c nude mice bearing either subcutaneous Capan-1 tumors, or intracranial Capan-1-luc tumors were dosed orally for up to 50 days with Niraparib (15, 30, or 45 mg/kg qd) or Olaparib (75 mg/kg qd). Tumor growth was monitored by either weekly physical measurement or quantification of bioluminescent signal in subcutaneous and intracranial models, respectively. Results Concentration-time profiles of niraparib in the brain were similar to those observed in the plasma after oral gavage dosing in rats, with an increase in Tmax observed for brain concentrations. Mean brain-to-plasma concentration ratios for niraparib following a single oral dose to rats were 0.85-0.99 of the brain Tmax. After a single oral dose, brain Ctrough levels (24hrs) were 2-4 times greater than observed in plasma, indicating niraparib is able to penetrate the brain in rodents. To evaluate activity in the brain response to niraparib was determined in a single xenograft model implanted either subcutaneously or intracranially. In the subcutaneous model, niraparib at 15, 30, or 45 mg/kg produced antitumor activity with tumor growth inhibition (TGI) values of 13, 49, or 54%, respectively (p Conclusions These data indicate that niraparib has sufficient exposure in rodent brains to have therapeutic benefit in an intracranial BRCA-mutant human xenograft model shown to be moderately sensitive to PARP inhibition. These results support the clinical investigation of niraparib against BRCAmut tumors that have metastasized to the brain. Citation Format: Keith Mikule, Keith Wilcoxen. The PARP inhibitor, niraparib, crosses the blood brain barrier in rodents and is efficacious in a BRCA2-mutant intracranial tumor model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B168.

Abstract LB-253: Inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis

Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug-senstivity, and their potential to metastasize and cause relapse. Subpopulations of cancer cells with extremely high tumorigenic potential have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties termed cancer stem cells. These stemness-high cancer cells are extremely tumorigenic and are resistant to conventional therapeutics due to activation of pro-survival and anti-apoptotic pathways, overexpression of drug efflux pumps, and increased DNA repair capacity. Moreover, chemotherapy and radiation have been found to induce stemness genes in cancer cells, converting stemness-low cancer cells to stemness-high cancer cells. Such highly tumorigenic and drug-resistant stemness-high cancer stem cells are, therefore, likely to be “left-over” following chemotherapy or radiotherapy and ultimately responsible for relapse. We hypothesized that cancer stemness inhibition is sufficient to suppress metastasis and relapse. Stemness, initially defined by the expression of stem cells genes, is a property shared by embryonic stem cells and adult stem cells. It has been demonstrated that the gene expression profiles of cancer stem cells more closely resemble embryonic stem cells than adult stem cells, suggesting the feasibility to identify molecular targets that are required for cancer stemness, but not (or less so) by normal adult stem cells. Through gene-silencing approaches, we have identified Stat3 as critically important for maintaining cancer stemness, yet largely dispensable for adult stem cells. Here we show that BBI608, a small molecule identified by its ability to inhibit gene-transcription driven by Stat3 and cancer cell stemness properties, displays anticancer properties that are highly different from chemotherapeutics agents. Stemness-high cancer cells enriched by multiple techniques are resistance to chemotherapeutics, yet highly sensitive to the stemness inhibitor BBI608. Blockade of spherogenesis and reduction of stemness gene expression by BBI608 were observed in stemness-high cancer cells isolated from a variety of cancer types. While treatment of xenografted tumor models with chemotherapeutics enriched stemness-high cancer cells, BBI608 induced significant depletion of stemness-high populations in vivo. Moreover, the inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis in xenografted human cancers in mice. These data demonstrate targeting cancer stemness as an effective way to suppress cancer relapse and metastasis. Citation Format: Youzhi Li, Harry A. Rogoff, Sarah Keates, Yuan Gao, Sylaja Murikipudi, Keith Mikule, David Leggett, Wei Li, Arthur Pardee, Chiang J. Li. Inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis. [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 LB-253. doi:10.1158/1538-7445.AM2015-LB-253

Abstract A221: Novel PARP6 inhibitors demonstrate in vivo efficacy in xenograft models.

The poly (ADP-ribose) polymerase (PARP) family of enzymes are functionally implicated in DNA repair, transcriptional regulation, glucose metabolism, mitosis, and other cellular mechanisms. To date, the focus has been on defining the role of PARPs 1-3 in DNA damage repair. Currently, PARP1-3 inhibitors are in clinical trials for BRCA1/2 mutant ovarian and breast cancers. The roles of other PARP family members are starting to emerge. Using a cell-based assay measuring multi-polar spindle induction, PARP6 was identified as novel target with therapeutic potential. AZ482 and AZ108 were developed as potent PARP6 inhibitors. These compounds exhibited a unique and selective growth inhibition profile when screened in large panels of tumor cells and suitable pharmacokinetics in vivo. In sensitive hematological and solid tumor models, these compounds had low nanomolar potency in vitro and anti-tumor efficacy in vivo. Mechanistic studies suggest PARP6 inhibitors disrupt spindle pole clustering resulting in mitotic catastrophe and may show selectivity toward tumor cell lines with supernumary centrosomes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A221. Citation Format: Michele F. Mayo, Shaun Grosskurth, Xin Wang, Philip Petteruti, Prasad Nadella, Corinne Reimer, Keith Mikule. Novel PARP6 inhibitors demonstrate in vivo efficacy in xenograft models. [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 A221.

Abstract A134: Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines.

Poly(ADP-ribose) polymerases (PARP) are a family of 17 enzymes that catalyze the transfer of the ADP-ribose from NADH to post-translationally modify (PTM) acceptor proteins. Currently, the PTM regulation of proteins by PARPs have been demonstrated to regulate numerous signaling cascades including but not limited to DNA damage response and tumor development for PARP1-3 as well as telomere maintenance, spindle assembly, vesicular movement, and regulation of beta-catenin destruction complex for PARP5a-5b. Although progress on the biological role and therapeutic potential for some PARPs has been made, the function of and possible therapeutic application for other PARPs are not fully understood. Using cell free enzyme and cell-base multi-polar spindle assays, semi-selective PARP6 inhibitors AZ482 and AZ108 were identified. From in vitro PARP6 knock-down and AZ108 pharmacological studies in breast cancer cell lines, PARP6 perturbation was demonstrated to disrupt spindle pole clustering. To identify PARP6 protein substrate that may contribute to the spindle pole clustering defect, an in vitro acceptor protein substrate screen was performed using high-density protein microarrays containing more than 8,000 unique proteins. From this screen, an enrichment for centromeric and microtubule organizing proteins was identified. Of these centromeric PARP6 protein substrate, CHEK1 and CENP were shown to be modulated by AZ108 treatment in breast cancer cell lines. These results implicate PARP6 in a role for stablization of the spindle assembly during mitosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A134. Citation Format: SHAUN GROSSKURTH, Philip Petteruti, Xin Wang, Keith Mikule. Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines. [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 A134.

Abstract 1724: Evaluation of niraparib in combination with anti-PD1/anti-PD-L1 in preclinical models

Niraparib is an orally available and selective poly(ADP-ribose) polymerase (PARP)-1/-2 inhibitor approved for maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in complete or partial response to platinum-based chemotherapy. PARP inhibition may enhance the immune response in tumors treated with anti-PD-1 therapy via generation of cytosolic DNA that activates T cells through the stimulator of interferon gene (STING) pathway, rendering tumors immunologically “hot” with an increase in infiltrating lymphocytes. In this study, we explored the responses and mechanism of action of niraparib and anti-PD-1/anti-PD-L1 combination therapy in preclinical models. Out of a cohort of 14 immune-competent mouse tumor models, the combination treatment demonstrated enhanced anti-tumor activity in eight tumor models derived from BRCA-proficient and BRCA-deficient genetic backgrounds. Substantial increases compared to monotherapies in anti-tumor activity was observed in 5 models, indicative of synergy between niraparib and anti-PD-1/anti-PD-L1 therapy. The combination triggered durable responses that were coincident with induction of immune memories in a BRCA-deficient ovarian syngeneic model. Mechanistically, niraparib treatment increased the number of infiltrating CD8+ and CD4+ cells within the intratumoral region. The enhanced immune cell infiltration was accompanied by elevated interferon-stimulated gene expression. Pathway analyses using transcriptome profiling identified interferon response gene signatures as the significantly differentially-upregulated gene sets following niraparib treatment. Consistently, niraparib treatment activated the STING pathway in vitro in BRCA-deficient MDA-MB-436 human triple negative breast cancer cells. STING pathway markers including p-STING(Ser366), p-TBK1(Ser172) and p-NFκB p65 were elevated following niraparib treatment and was accompanied by an increase in IFNB mRNA expression. In summary, our data suggested that niraparib treatment in combination with anti-PD-1/anti-PD-L1 therapy enhanced immune cell infiltration, interferon-stimulated gene expression and tumor responses. Citation Format: Sarah Wang, Kaiming Sun, Yonghong Xiao, Bin Feng, Keith Mikule, Sridhar Ramaswamy, Jeffrey Hanke, Jing Wang. Evaluation of niraparib in combination with anti-PD1/anti-PD-L1 in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1724.

Abstract 4353: Preclinical evaluation of the PARP inhibitor niraparib and cytotoxic chemotherapy alone or in combination in a panel of 25 triple-negative breast cancer PDX models: Relevance of BRCA mutations, HRD status and other biomarkers

Poly-ADP-ribose polymerase 1 (PARP) inhibitors are therapeutically effective in a subset of breast and high-grade ovarian cancers harboring deleterious BRCA1/2 mutations. Recently, several studies have tried to identify other molecular markers of HRD in BRCA1/2 wild-type (wt) breast and ovarian cancers to identify patients that could benefit from PARP inhibitor treatment. Most clinical studies of single-agent PARP inhibitors are therefore focused on breast or ovarian cancer with BRCA1/2 mutations and/or identified as HRD. Preclinical evidence also indicates that PARP inhibitors have therapeutic potential in combination with genotoxic chemotherapy. However, the toxicity associated with such combinations has generally imposed dose reduction of the chemotherapy, possibly explaining why the clinical activity observed with the combinations has in general not been superior to that of the corresponding single agents given at full dose. In a panel of 25 triple negative breast cancer (TNBC) PDX models, we investigated the antitumor efficacy of the PARP inhibitor niraparib used alone or in combination with cyclophosphamide, a backbone genotoxic drug of TNBC standard chemotherapy. Responses were correlated with both BRCA1/2 mutations and HRD status as defined by the myChoice HRD™ test from Myriad Genetics and with other HRD markers. Responses to cyclophosphamide ranged from progression to partial or complete tumor regression. Responses to niraparib were also variable and occurred only in tumors with BRCA mutations or a high HRD score. High sensitivities to niraparib and cyclophosphamide were tightly associated, suggesting that they may both be a result of a defect in a BRCA1/2-related pathway. We tested if niraparib could potentiate the efficacy of cyclophosphamide in TNBC models that initially regressed on cyclophosphamide but eventually relapsed. We chose a gapped sequential design in which treatment with niraparib was initiated 14 days after a full dose of cyclophosphamide, to avoid toxicity associated with concomitant administration. Potentiation with inhibition of tumor relapse was observed in tumors sensitive to single-agent niraparib, but not in tumors refractory to single-agent niraparib. In responder tumors, the cyclophosphamide-niraparib sequential combination was superior to sequential cycles of cyclophosphamide in preventing tumor relapse. These results show that a gapped sequential combination of full dose cyclophosphamide and niraparib is well tolerated and demonstrates remarkable efficacy in a subset of TNBCs. The data will be discussed with regard to potential adjuvant treatment strategies with PARP inhibitors in TNBC and the value of myChoice HRD™ as a biomarker in predicting response to PARP inhibition alone and in combination with chemotherapy. Citation Format: Olivier Deas, Stefano Cairo, Keith Wilcoxen, Keith Mikule, Truong-An Tran, Kirsten Timms, Jean-Gabriel Judde. Preclinical evaluation of the PARP inhibitor niraparib and cytotoxic chemotherapy alone or in combination in a panel of 25 triple-negative breast cancer PDX models: Relevance of BRCA mutations, HRD status and other biomarkers. [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 4353.