Abstract: R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes, including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell–enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo. Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among patients with breast cancer. Thus, we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers. Significance: These findings identify RSPO3 as a potential therapetuic target in basal-like breast cancers. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4497/F1.large.jpg. Cancer Res; 78(16); 4497–511. ©2018 AACR.

Abstract: Purpose: According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. Experimental Design: We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. Results: We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. Conclusion: We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. Clin Cancer Res; 20(4); 1007–19. ©2013 AACR.

Abstract: MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells. Cancer Res; 70(24); 10255–64. ©2010 AACR.

Abstract: The tyrosine threonine kinase TTK, also known as Monopolar Spindle 1 kinase (MPS1), is a conserved kinase found to be highly expressed in a number of human tumors of different origin. TTK kinase activity plays a critical role in the control of mitosis, regulating the spindle assembly checkpoint (SAC), a mitotic mechanism required for proper chromosome alignment and segregation during cellular division. The activity of this checkpoint has been shown to be proficient and up-regulated in aneuploid tumors, a common feature measured in approximately 90% of solid tumors and 70% of hematological cancers. S81694 (NMS-P153) is a novel potent inhibitor of TTK. It is a tight binder compound with long residence time on the target and is highly selective for TTK against a wide range of tested enzymes (kinases, channels, hormones, GPCRs). In vitro, brief exposure is sufficient to commit cells to death with the compound showing anti-proliferative activity with a demonstrated mechanism of action and marked tumor growth inhibition in preclinical in vivo tumor models associated with a good pharmacokinetic profile in both rodent and non-rodent species. TTK inhibitors are anticipated to be more active in SAC dependent tumors and data in the literature suggests that triple negative breast cancer is among the tumor types potentially highly dependent on TTK activity. Moreover, in accelerating mitosis, TTK inhibitors have a novel mode of action as compared to currently known mitotic inhibitors. To confirm the role of TTK in breast cancer and identify a sensitive patient population to S81694 treatment, translational studies have been conducted on a large panel of human tumor cell lines, including 39 of mammary gland origin. Anti-proliferative activity was measured after 72h continuous treatment or after a brief treatment of 7h followed by 2D colony forming assay to verify the long term effects of a treatment which mimics compound half-life in mice. In addition, active caspase 3 induction was also analyzed to identify cell lines more prone to activate apoptosis upon treatment. Triple negative breast cancer (TNBC) cell lines were confirmed to be particularly sensitive to S81694 and correlation was observed between sensitivity profile in vitro and tumor growth inhibition in vivo. In MDA-MB-231 xenograft and orthotopic models, tumor regression, cured animals and metastasis reduction in several organs including brain and lungs was observed. These results further confirm the role of TTK in triple negative breast cancer and could support the clinical development of S81694 in this indication. Citation Format: Riccardo Colombo, Mike Burbridge, Marianne Rodriguez, Frédérique Cantero, Marina Caldarelli, Maria Laura Giorgini, Francesco Sola, Dario Ballinari, Marina Ciomei, Roberta Bosotti, Alessia Montagnoli, Antonella Isacchi, Daniele Donati, Arturo Galvani. Preclinical characterization of the novel TTK kinase inhibitor S81694 for the treatment of triple negative breast cancer. [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 1638. doi:10.1158/1538-7445.AM2015-1638

Abstract: Purpose: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy. Experimental Design: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer. Results: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632. Conclusions: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.

Abstract: Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase considered to be the master player of cell-cycle regulation during mitosis. It is indeed involved in centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 is overexpressed in a variety of human tumors and its overexpression often correlates with poor prognosis. Although five different PLKs are described in humans, depletion or inhibition of kinase activity of PLK1 is sufficient to induce cell-cycle arrest and apoptosis in cancer cell lines and in xenograft tumor models. NMS-P937 is a novel, orally available PLK1-specific inhibitor. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid and hematologic tumors. NMS-P937 potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with clear PLK1-related mechanism of action at well-tolerated doses in mice after oral administration. In addition, NMS-P937 shows potential for combination in clinical settings with approved cytotoxic drugs, causing tumor regression in HT29 human colon adenocarcinoma xenografts upon combination with irinotecan and prolonged survival of animals in a disseminated model of acute myelogenous leukemia in combination with cytarabine. NMS-P937, with its favorable pharmacologic parameters, good oral bioavailability in rodent and nonrodent species, and proven antitumor activity in different preclinical models using a variety of dosing regimens, potentially provides a high degree of flexibility in dosing schedules and warrants investigation in clinical settings. Mol Cancer Ther; 11(4); 1006–16. ©2012 AACR.

Abstract: Aneuploidy is one of the major hallmarks of cancer and is a unique property of tumoral cells. Thus, targeting the molecular mechanisms required for the growth of aneuploid cells might be expected to yield more cancer cell specific therapeutic approaches. The Spindle Assembly Checkpoint (SAC) is a mitotic mechanism required for proper chromosomal segregation, ensuring that cells do not divide until all sister chromatids correctly align to the metaphase plate. It is a complex machinery tightly regulated by a number of members which are rarely mutated in tumors. Rather, checkpoint components have been found to be upregulated in aneuploid tumor cells, suggesting a potential tumor “addiction” to SAC functions. MPS1 kinase, (also known as TTK) is an essential SAC enzyme aberrantly overexpressed in a wide range of tumors and necessary for tumor cell proliferation. We previously reported the identification of NMS-P715 as a selective and orally bioavailable MPS1 inhibitor capable of promoting mitotic acceleration, thereby causing aneuploidy and cancer cell death both in vitro and in vivo. Supported by the proof that MPS1 small-molecule inhibitors display the expected mechanism of action and promote tumor growth inhibition in preclinical cancer models, further medicinal chemistry efforts were undertaken and led to the discovery of NMS-P153 as an early candidate product. NMS-P153 is highly selective, and compared to NMS-P715, shows improved in vitro enzymatic potency on MPS1 (IC50 of 3 nM), 10-fold increased mitotic override activity, longer dissociation half-life and better mouse and rat PK parameters, leading to impressive tumor growth inhibition associated with tumor regression in efficacy studies. Proliferation data performed on a large panel of cell lines ( & gt;100) have been obtained and will be essential to decipher the most sensitive genetic backgrounds to chemical abrogation of MPS1 function. In addition, single and repeated toxicology studies in rat are being carried out to establish therapeutic safety margins. Inhibiting the spindle assembly checkpoint by targeting MPS1 kinase could represent a promising new approach to cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B241.

Abstract: Most of the widely used anticancer drugs target the elongation step of DNA synthesis, either directly, for example by restricting the pool of dNTPs or by acting as chain terminators following incorporation into the nascent DNA strands, or indirectly, for example by targeting enzymes that facilitate replication fork progression, or by intercalating DNA and thereby creating a physical block to fork progression. The arrest of replication forks induced by these agents invariably results in DNA strand breakage, which, while being potentially toxic to normal proliferating tissues, is also susceptible to escape by tumor cells via activation of the ATR/ATM dependent S-phase checkpoint pathway and subsequent repair of the damaged lesion. Targeting the initiation step of DNA replication, rather than elongation, might represent a strategy to overcome activation of the S-phase checkpoint, and in this context a potentially attractive molecular target is the serine/threonine kinase Cdc-7. Cdc7 promotes DNA replication initiation via phosphorylation of one or more subunits of the MCM DNA helicase complex, thus facilitating unwinding of double-stranded DNA at replication origins. Phosphorylation of Mcm2 at Ser40 and Ser53 is uniquely dependent upon Cdc7 and is essential for initiation of DNA replication and cell growth. The altered expression of proteins involved in the initiation of DNA replication closely correlates with aggressive tumor phenotypes and is a powerful marker of clinical outcome in a variety of malignancies. In particular, Cdc7 protein levels and Cdc7 dependent activity are increased in many cancer cell lines, and in tumors compared to matched normal tissue, where correlation with negative prognosis has been observed for ovarian, breast, colon and uterine carcinomas, diffuse large B cell non-Hodgkin's and Hodgkin's lymphomas. Somatic mutations of the CDC7 gene have also been identified in colorectal and gastric carcinomas. Depletion of Cdc7 by RNA interference results in p53 independent apoptosis of tumor cells, whereas normal cells respond with reversible arrest of cell cycle progression, perhaps corresponding to a physiological checkpoint. Sustained inhibition of Cdc7 in the presence of drugs affecting the elongation step of DNA replication such as topoisomerase inhibitors or DNA intercalating agents, increases cell death. Taken together, these findings suggest Cdc7 kinase may represent a promising novel cancer target. NMS-1116354 is a potent (low nM) ATP competitive small molecule inhibitor of Cdc7 kinase activity. Tested on a panel of more than 50 kinases representative of the human kinome, NMS-1116354 was found to be highly selective, with cross-reactivity observed only for CDK9. NMS-1116354 was tested for anti-proliferative activity against a panel of 171 human cancer cell lines of both solid and hematological origin. Cancer cell proliferation was inhibited with IC50 values & lt; 1 μM in 99 cell lines. Lymphoma, multiple myeloma and breast cancer cell lines, in particular triple negative, were especially sensitive to NMS-1116354. Activity of NMS-1116354 was independent of p53 status, and was maintained in cell lines resistant to 5-FU, cisplatin, gemcitabine and doxorubicin. NMS-1116354 induced rapid (within 3 hours of treatment) and massive cell apoptosis as assessed by induction of active caspase 3 and sub-G1 accumulation. Thus, in vitro, NMS-1116354 has potent, broad and p53-independent anti-cancer activity. In mechanism of action studies in cells, NMS-1116354 inhibited pSer40-Mcm2 (a phosphorylation event which we have previously described as being a specific biomarker of Cdc7 kinase activity) at drug concentrations consistent with those required for cell proliferation inhibition and apoptosis induction. Unlike DNA elongation inhibitors, such as hydroxyurea (HU), NMS-1116354 did not induce the DNA damage checkpoint response as assessed by Chk1 and Chk2 phosphorylation. Thus, effects observed following treatment of cells with NMS-1116354 essentially recapitulated those observed after genetic ablation of the protein using anti-Cdc7 siRNA. In cells, NMS-1116354 was also found to down-regulate the pro-survival protein Mcl-1, consistent with inhibition of Cdk9, the only other tested kinase against which the compound has significant activity. Downregulation of Mcl-1 protein may thus potentially contribute to anti-tumor activity of the compound, particularly in contexts which are dependent on the anti-apoptotic activity of this protein. NMS-1116354 has excellent oral biovailalability and is well tolerated in mice after prolonged treatment. Administration of NMS-1116354 to tumor-bearing animals induced potent tumor growth inhibition, including tumor regression, in xenograft models of human breast, colon, and ovarian cancer, as well as in the transgenic TRAMP model of prostate carcinoma. Tumor regression in 7 out of 10 treated animals was also observed in the rat DMBA carcinogen induced mammary tumor model. NMS-1116354 treatment also increased survival time and induced tumor regressions in AML and multiple myeloma animal models. Ex-vivo analysis of mouse xenografts treated with NMS-1116354 showed dose dependent inhibition of Mcm2 phosphorylation and modulation of the expression of a specific set of Cdc7 regulated genes in skin and tumors after both single and repeated administration. Additionally, Mcl-1 expression was found to be down-regulated in white blood cells of treated mice, as well as in tumor cells in the case of hematological cancer models, where interestingly, down-regulation correlated with antitumor activity. Thus, NMS-1116354 has potent anti-cancer activity in vivo on several different solid and hematological cancer models, in which the compound inhibits Cdc7 kinase activity and reduces Mcl-1 protein levels. These findings confirm the dual mechanism of action of the compound in vivo and support the use of Mcm2 phosphorylation, Mcl-1 down-regulation and a specifically identified gene signature as biomarkers of target modulation in clinical trials, both in tumors and in surrogate tissues (i.e. skin and blood). In drug combination studies, NMS-1116354 exhibited synergistic effects when combined with Irinotecan, Docetaxel, anti-metabolites (Gemcitabine and 5-FU) and Bortezomib in vitro and in vivo, opening a possible path for its clinical development in combination with approved drugs. Phase I clinical trials to evaluate the safety of NMS-1116354 in cancer patients were initiated in 2009. The Phase I program currently ongoing includes clinical studies with NMS-1116354 administered orally as single agent exploring different schedules in patients with solid tumors and hematological malignancies. In conclusion, NMS-1116354 is a novel Cdc7 and Cdk9 kinase inhibitor with a unique mechanism of action which involves inhibition of initiation of DNA replication and down-regulation of the pro-survival protein Mcl-1. The compound is endowed with potent single agent antitumor activity in solid and hematological cancer models, is synergistic in combination with approved drugs, and is presently in Phase I clinical studies. Selected References Montagnoli A. et al., Drf1, a novel regulatory subunit for human Cdc7 kinase. EMBO J. 2002; 21: 3171-3181. Montagnoli A. et al., Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells. Cancer Res. 2004; 64: 7110-7116. Montagnoli A. et al., Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases. J Biol. Chem. 2006; 281: 10281-10290. Tenca P. et al., Cdc7 is an active kinase in human cancer cells undergoing replication stress. J Biol. Chem. 2007; 282: 208-215. Montagnoli A. et al., A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nature Chem. Biol. 2008; 4: 357-365. Vanotti E. et al., Cdc7 kinase inhibitors: pyrrolopyridinones as potential antitumor agents. Synthesis and structure-activity relationships. J. Med. Chem. 2008; 51: 487-501. Menichincheri et al., First Cdc7 Kinase Inhibitors: Pyrrolopyridinones as Potent and Orally Active Antitumor Agents. 2. Lead Discovery. J. Med. Chem. 2009, 22; 293-307. Ermoli et al. Cell division cycle 7 kinase inhibitors: 1H-pyrrolopyridines, synthesis and structure-activity relationships. J. Med. Chem. 2009, 23; 4380-90. Warr MR, Shore GC., Unique biology of Mcl-1: therapeutic opportunities in cancer. Current Molecular Medicine 2008; 8: 138-147. Citation Format: Francesco Colotta, Jurgen Moll, Barbara Valsasina, Ermes Vanotti, Sonia Rainoldi, Francesco Sola, Vanessa Marchesi, Maria Menichincheri, Antonella Ciavolella, Veronica Patton, Clara Albanese, Daniele Volpi, Nilla Avanzi, Dario Ballinari, Francesco Fiorentini, Antonella Isacchi, Enrico Pesenti, Arturo Galvani, Corrado Santocanale, Alessia Montagnoli. NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-01

Abstract: Background: People with HIV (PWH) have a higher risk of acquiring oral HPV infection when compared to people without HIV, which can lead to the high prevalence of oropharyngeal cancers in PWH. Puerto Rico (PR), a US jurisdiction, is on the top 10 in terms of HIV prevalence and cumulative AIDS cases among the US and have a significant burden and disparity of HPV-related malignancies. Additionally, PR has socio-economic disadvantages, which is a social determinant of oral health disparities, and can potentially affect oral HPV. The relationship of these social determinants with HPV have not been investigated in PWH in PR. Hence, in this study, we evaluated the relationship between socio-economic, behavioral, and oral health factors with oral HPV among PWH. Methods: We evaluated 117 sexually active PWH (102 males and 15 females) from a community clinic in San Juan, PR. Oral rinse samples were collected and analyzed for HPV infection and genotype using the DNA ELISA kit HPV SPF10 and RHA kit HPV SPF10-LiPA25. We collected sociodemographic characteristics (age, marital status, employment, income) as well as lifestyle variables (smoking habits, alcohol consumption, sexual practices) through an administered questionnaire. Participants also underwent a clinical full-mouth periodontal assessment following the CDC/AAP guidelines. Statistical analyses in relation to HPV were performed in R statistical software. Findings: There was a prevalence of oral HPV infection of 29.9% in PWH in PR, which 70% were high-risk genotypes, with HPV-18 being the most abundant genotype (27.5%). There were 33.3% of males who had oral HPV while only 20% of females had oral HPV. Also, oral HPV infection was significantly associated with periodontitis (p & lt;0.05). In men, oral HPV positivity trended to be frequent with no HPV vaccination (p=0.08). No HPV vaccination was associated with lower income (p & lt;0.05). There was no association between HPV status and sociodemographic and lifestyle variables. Interpretation: The prevalence in high-risk HPV genotypes among PWH was higher than what has previously been observed in PR. We also found a higher prevalence of HPV-18, which may explain clinical disparities concerning oropharyngeal cancer in PR’s population. Additionally, our findings highlight the importance of access to medical care in low-resource settings. Low income can be a surrogate of lack of access of care, which can impact early diagnosis, risk, and prevention of oral HPV. Therefore, targeting social determinants, may help lower the risk of infection and oral cancer in underserved populations. While our findings contribute to understanding the impact of social determinants in oral cancer, more studies are still needed that investigate the relationship between other sociodemographic factors that affect oral health in PWH. Citation Format: Carlos A. Solá-Morlá, Yabdiel Ramos-Valerio, Coral Martes-Villalobos, María M. Sánchez-Vázquez, Ramón F. Gonzalez-García, Ana P. Ortiz, Magaly Martinez-Ferrer, Josué Pérez-Santiago. Social determinants associated with HPV disparities among people with HIV infection in Puerto Rico [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B130.

Abstract: The Spindle Assembly Checkpoint (SAC) is a mitotic mechanism specifically required for proper chromosomal segregation ensuring that cells do not divide until all sister chromatids correctly align to the metaphase plate. MPS1 kinase, (also known as TTK) is a key regulator of SAC functions and it has been found to be up-regulated in a number of tumors of different origins. The hypothesis is that SAC activity could be highly required to sustain aneuploid tumor growth and MPS1 inhibitors may have a therapeutic benefit in the treatment of certain cancers. We have identified NMS-P715, a potent and selective oral bioavailable small-molecule MPS1 kinase inhibitor with an ATP-competitive bindingmode. Treatment of cells with NMS-P715 accelerates mitotic exit with an IC50 of 53 nM. This is accompanied by reduction of the mitotic length, MPS1 dephosphorylation, chromosomal mis-alignment, delocalization of kinetochore components and massive aneuploidization, which ultimately leads to cell death. Proliferation data performed on a large panel of 126 cell lines shows a wide range of activity and indicates selective activity against tumoral cells compared to normal cells. Oral administration of NMS-P715 in tumor xenografted mice resulted in potent tumor growth inhibition in an ovarian cancer xenograft model and was accompanied by biomarker modulation, confirming the expected MOA. Our data provide evidence that inhibition of MPS1 kinase and SAC abrogation could represent a new promising approach to cancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C202.