KDM6A Loss Triggers an Epigenetic Switch That Disrupts Urothelial Differentiation and Drives Cell Proliferation in Bladder Cancer.

Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability. SIGNIFICANCE: A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

Rare germline alterations of myeloperoxidase predispose to myeloid neoplasms.

Myeloperoxidase (MPO) gene alterations with variable clinical penetrance have been found in hereditary MPO deficiency, but their leukemia association in patients and carriers has not been established. Germline MPO alterations were found to be significantly enriched in myeloid neoplasms: 28 pathogenic/likely pathogenic variants were identified in 100 patients. The most common alterations were c.2031-2 A > C, R569W, M519fs* and Y173C accounting for about half of the cases. While functional experiments showed that the marrow stem cell pool of Mpo-/- mice was not increased, using competitive repopulation demonstrated that Mpo-/- grafts gained growth advantage over MPO wild type cells. This finding also correlated with increased clonogenic potential after serial replating in the setting of H2O2-induced oxidative stress. Furthermore, we demonstrated that H2O2-induced DNA damage and activation of error-prone DNA repair may result in secondary genetic damage potentially predisposing to leukemia leukemic evolution. In conclusion, our study for the first time demonstrates that germline MPO variants may constitute risk alleles for MN evolution.

Eltrombopag inhibits TET dioxygenase to contribute to hematopoietic stem cell expansion in aplastic anemia.

Eltrombopag, an FDA-approved non-peptidyl thrombopoietin receptor agonist, is clinically used for the treatment of aplastic anemia, a disease characterized by hematopoietic stem cell failure and pancytopenia, to improve platelet counts and stem cell function. Eltrombopag treatment results in a durable trilineage hematopoietic expansion in patients. Some of the eltrombopag hematopoietic activity has been attributed to its off-target effects, including iron chelation properties. However, the mechanism of action for its full spectrum of clinical effects is still poorly understood. Here, we report that eltrombopag bound to the TET2 catalytic domain and inhibited its dioxygenase activity, which was independent of its role as an iron chelator. The DNA demethylating enzyme TET2, essential for hematopoietic stem cell differentiation and lineage commitment, is frequently mutated in myeloid malignancies. Eltrombopag treatment expanded TET2-proficient normal hematopoietic stem and progenitor cells, in part because of its ability to mimic loss of TET2 with simultaneous thrombopoietin receptor activation. On the contrary, TET inhibition in TET2 mutant malignant myeloid cells prevented neoplastic clonal evolution in vitro and in vivo. This mechanism of action may offer a restorative therapeutic index and provide a scientific rationale to treat selected patients with TET2 mutant-associated or TET deficiency-associated myeloid malignancies.

PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation.

PBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.

Therapeutic Targeting of Protein Disulfide Isomerase PDIA1 in Multiple Myeloma.

Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in myeloma.

A Therapeutic Strategy for Preferential Targeting of TET2 Mutant and TET-dioxygenase Deficient Cells in Myeloid Neoplasms.

TET2 is frequently mutated in myeloid neoplasms. Genetic TET2 deficiency leads to skewed myeloid differentiation and clonal expansion, but minimal residual TET activity is critical for survival of neoplastic progenitor and stem cells. Consistent with mutual exclusivity of TET2 and neomorphic IDH1/2 mutations, here we report that IDH1/2 mutant-derived 2-hydroxyglutarate is synthetically lethal to TET-dioxygenase deficient cells. In addition, a TET-selective small molecule inhibitor decreased cytosine hydroxymethylation and restricted clonal outgrowth of TET2 mutant, but not normal hematopoietic precursor cells in vitro and in vivo. While TET-inhibitor phenocopied somatic TET2 mutations, its pharmacologic effects on normal stem cells were, unlike mutations, reversible. Treatment with TET inhibitor suppressed the clonal evolution of TET2 mutant cells in murine models and TET2-mutated human leukemia xenografts. These results suggest that TET inhibitors may constitute a new class of targeted agents in TET2 mutant neoplasia.

CBL0137 increases the targeting efficacy of Rovalpituzumab tesirine against tumour-initiating cells in small cell lung cancer.

Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody-drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients.

Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma.

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.

PRO 140 Monoclonal Antibody to CCR5 Prevents Acute Xenogeneic Graft-versus-Host Disease in NOD-scid IL-2Rynull Mice.

Graft-versus-host disease (GVHD) is a prevalent and potentially lethal complication of hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic GVHD are important tools used to study the human immune response in vivo. Here we used NOD-scid IL-2Rynull mice (NSG) transplanted with human bone marrow stem cells to evaluate the role of immune cell engraftment in the production of acute GVHD. PRO 140, a humanized monoclonal antibody targeting the chemokine receptor, CCR5, was used to evaluate its influence on bone marrow cell engraftment and modulation of acute GVHD. We evaluated the kinetics of engraftment by determining the percentage and absolute numbers of human CD45+ cells and CD3+ T cells from peripheral blood, spleen, and bone marrow in treated and control mice. With a dosing schedule of 2 mg of test or control antibody administered i.p. twice weekly, PRO 140-treated mice showed no signs of GVHD throughout the 70-day study period and gained weight until they were killed at 70 days for flow cytometry analysis. Control mice started losing weight after 25 days, showed classic signs of GVHD (ruffled fur, lethargy, severe hunching), and all were killed by day 54. The percentage and absolute numbers of human CD45+ cells in peripheral blood increased in both groups of mice throughout the 50-day comparison period and was lower in the PRO 140-treated mice at day 50. There was no difference in human CD45+ cells detected in bone marrow from control and PRO 140-treated killed mice. At this time point 76.1% and 68.2% of the hematopoietic cells from peripheral blood and from bone marrow, respectively, were of human lineage and 14.9% and 28%, respectively, were of mouse origin. With a schedule using 10-fold less dose of antibody (.2 mg i.p. twice weekly), PRO 140 still significantly modulated acute GVHD in terms of both weight loss and survival times, but no mice from either control or test group survived. By targeting the CCR5 chemokine receptor, PRO 140 modulated acute GVHD in a dose-response fashion in this xenogeneic mouse model without significantly altering engraftment.

CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors.

Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

Kindlin-2 Regulates the Growth of Breast Cancer Tumors by Activating CSF-1-Mediated Macrophage Infiltration.

Interplay between tumor cells and host cells in the tumor microenvironment dictates the development of all cancers. In breast cancer, malignant cells educate host macrophages to adopt a protumorigenic phenotype. In this study, we show how the integrin-regulatory protein kindlin-2 (FERMT2) promotes metastatic progression of breast cancer through the recruitment and subversion of host macrophages. Kindlin-2 expression was elevated in breast cancer biopsy tissues where its levels correlated with reduced patient survival. On the basis of these observations, we used CRISPR/Cas9 technology to ablate Kindlin-2 expression in human MDA-MB-231 and murine 4T1 breast cancer cells. Kindlin-2 deficiency inhibited invasive and migratory properties in vitro without affecting proliferation rates. However, in vivo tumor outgrowth was inhibited by >80% in a manner associated with reduced macrophage infiltration and secretion of the macrophage attractant and growth factor colony-stimulating factor-1 (CSF-1). The observed loss of CSF-1 appeared to be caused by a more proximal deficiency in TGFß-dependent signaling in Kindlin-2-deficient cells. Collectively, our results illuminate a Kindlin-2/TGFß/CSF-1 signaling axis employed by breast cancer cells to capture host macrophage functions that drive tumor progression. Cancer Res; 77(18); 5129-41. ©2017 AACR.

MEK inhibition abrogates sunitinib resistance in a renal cell carcinoma patient-derived xenograft model.

BACKGROUND: Renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKI) typically respond initially, but usually develop resistance to therapy. We utilised transcriptome analysis to identify gene expression changes during development of sunitinib resistance in a RCC patient-derived xenograft (PDX) model. METHODS: RCC tumours were harvested during pre-treatment, response and escape phases. Direct anti-proliferative effects of sunitinib plus MEK inhibitor were assessed. Activation status (phosphorylation) of MEK1/2 and ERK1/2 was determined, myeloid-derived suppressor cells (MDSC) sub-fractions were quantitated and G-CSF was measured by ELISA. RESULTS: During the response phase, tumours exhibited 91% reduction in volume, characterised by decreased expression of cell survival genes. After 4-week treatment, tumours developed resistance to sunitinib, associated with increased expression of pro-angiogenic and cell survival genes. During tumour escape, cellular movement, inflammatory response and immune cell trafficking genes were induced, along with intra-tumoural accumulation of MDSC. In this PDX model, either continuous treatment with sunitinib plus MEK inhibitor PD-0325901, or switching from sunitinib to PD-0325901 was effective. The combination of PD-0325901 with TKI suppressed intra-tumoural phospho-MEK1/2, phospho-ERK1/2 and MDSC. CONCLUSIONS: Continuous treatment with sunitinib alone did not maintain anti-tumour response; addition of MEK inhibitor abrogated resistance, leading to improved anti-tumour efficacy.

Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma.

Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow-sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340-50. ©2016 AACR.

RNase L is a negative regulator of cell migration.

RNase L is a regulated endoribonuclease that functions in the interferon antiviral response. Activation of RNase L by 2', 5'-oligoadenylates has been linked to apoptosis, autophagy and inflammation. Genetic studies have also suggested the possible involvement of the RNase L gene (RNASEL) on chromosome 1q25.3 in several types of cancer. Here we report that ablation of RNase L in human prostate cancer PC3 cells by CRISPR/Cas9 gene editing technology enhanced cell migration as determined both by transwell assays and scratch wound healing assays. In addition, RNase L knockdown by means of RNAi increased migration of PC3 and DU145 cells in response to either fibronectin or serum stimulation, as did homozygous disruption of the RNase L gene in mouse embryonic fibroblasts. Serum or fibronectin stimulation of focal adhesion kinase (FAK) autophosphorylation on tyrosine-397 was increased by either knockdown or ablation of RNase L. In contrast, a missense mutant RNase L (R667A) lacking catalytic activity failed to suppress cell migration in PC3 cells. However, a nuclease-inactive mutant mouse RNase L (W630A) was able to partially inhibit migration of mouse fibroblasts. Consistent with a role for the catalytic activity of RNase L, transfection of PC3 cells with the RNase L activator, 2', 5'-oligoadenylate, suppressed cell migration. RNase L knockdown in PC3 cells enhanced tumor growth and metastasis following implantation in the mouse prostate. Our results suggest that naturally occurring mutations in the RNase L gene might promote enhanced cell migration and metastasis.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms.

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

Splicing factor 3b subunit 1 (Sf3b1) haploinsufficient mice display features of low risk Myelodysplastic syndromes with ring sideroblasts.

BACKGROUND: The presence of somatic mutations in splicing factor 3b subunit 1 (SF3B1) in patients with Myelodysplastic syndromes with ring sideroblasts (MDS-RS) highlights the importance of the RNA-splicing machinery in MDS. We previously reported the presence of bone marrow (BM) RS in Sf3b1 heterozygous (Sf3b1 (+/-)) mice which are rarely found in mouse models of MDS. Sf3b1 (+/-) mice were originally engineered to study the interaction between polycomb genes and other proteins. METHODS: We used routine blood tests and histopathologic analysis of BM, spleen, and liver to evaluate the hematologic and morphologic characteristics of Sf3b1 (+/-) mice in the context of MDS by comparing the long term follow-up (15 months) of Sf3b1 (+/-) and Sf3b1 (+/+) mice. We then performed a comprehensive RNA-sequencing analysis to evaluate the transcriptome of BM cells from Sf3b1 (+/-) and Sf3b1 (+/+) mice. RESULTS: Sf3b1 (+/-) exhibited macrocytic anemia (MCV: 49.5 ± 1.6 vs 47.2 ± 1.4; Hgb: 5.5 ± 1.7 vs 7.2 ± 1.0) and thrombocytosis (PLTs: 911.4 ± 212.1 vs 878.4 ± 240.9) compared to Sf3b1 (+/+) mice. BM analysis showed dyserythropoiesis and occasional RS in Sf3b1 (+/-) mice. The splenic architecture showed increased megakaryocytes with hyperchromatic nuclei, and evidence of extramedullary hematopoiesis. RNA-sequencing showed higher expression of a gene set containing Jak2 in Sf3b1 (+/-) compared to Sf3b1 (+/+). CONCLUSIONS: Our study indicates that Sf3b1 (+/-) mice manifest features of low risk MDS-RS and may be relevant for preclinical therapeutic studies.

Synergistic simvastatin and metformin combination chemotherapy for osseous metastatic castration-resistant prostate cancer.

Docetaxel chemotherapy remains a standard of care for metastatic castration-resistant prostate cancer (CRPC). Docetaxel modestly increases survival, yet results in frequent occurrence of side effects and resistant disease. An alternate chemotherapy with greater efficacy and minimal side effects is needed. Acquisition of metabolic aberrations promoting increased survival and metastasis in CRPC cells includes constitutive activation of Akt, loss of adenosine monophosphate-activated protein kinase (AMPK) activity due to Ser-485/491 phosphorylation, and overexpression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoAR). We report that combination of simvastatin and metformin, within pharmacologic dose range (500 nmol/L to 4 µmol/L simvastatin and 250 µmol/L to 2 mmol/L metformin), significantly and synergistically reduces C4-2B3/B4 CRPC cell viability and metastatic properties, with minimal adverse effects on normal prostate epithelial cells. Combination of simvastatin and metformin decreased Akt Ser-473 and Thr-308 phosphorylation and AMPKα Ser-485/491 phosphorylation; increased Thr-172 phosphorylation and AMPKα activity, as assessed by increased Ser-79 and Ser-872 phosphorylation of acetyl-CoA carboxylase and HMG-CoAR, respectively; decreased HMG-CoAR activity; and reduced total cellular cholesterol and its synthesis in both cell lines. Studies of C4-2B4 orthotopic NCr-nu/nu mice further demonstrated that combination of simvastatin and metformin (3.5-7.0 µg/g body weight simvastatin and 175-350 µg/g body weight metformin) daily by oral gavage over a 9-week period significantly inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure more effectively than 24 µg/g body weight docetaxel intraperitoneally injected every 3 weeks, 7.0 µg/g/day simvastatin, or 350 µg/g/day metformin treatment alone, with significantly less toxicity and mortality than docetaxel, establishing combination of simvastatin and metformin as a promising chemotherapeutic alternative for metastatic CRPC.

The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects.

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.

The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell-cycle progression in malignant bladder cancer cells in vitro and in vivo.

PURPOSE: Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target. EXPERIMENTAL DESIGN: Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. RESULTS: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells. CONCLUSIONS: Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer.