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PDF file - 603K, Supplemental Figure S1: Gene maps of the BRCA1 (A) and BRCA2 (B) CpG islands examined with bisulfite pyrosequencing. Supplemental Figure S2: Anchorage independent growth inhibition at 1μM rucaparib in soft agar. Of the 39 tested ovarian cancer cell lines 22 formed colonies in soft agar. Supplemental Figure S3: Microarray hybridizations of the cell line panel were performed using the Agilent Whole Human Genome 4X44K array chip.Supplemental Figure S4: Chemical structures of compounds used for the drug interaction studies.Supplemental Figure S5: A multivariate ANOVA model (accounting for type of chemotherapy, cell line, and drug concentration as single factors) indicates that the drug combination effects differed significantly between individual chemotherapeutic agents (P=0.010). Supplemental Figure S6: A multivariate ANOVA model (accounting for type of chemotherapy, cell line, and drug concentration as single factors) indicates that the best combination effect was achieved at lower topotecan (0.031 μM) and rucaparib (0.625 μM) drug concentrations (P=0.018).

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. ©2013 AACR.

PDF file - 66K, Cell lines and culture media.

Supplementary Figure S1; Supplementary Table S1; Supplementary Tables S3-S4.

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer.Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients.Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052).Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Supplementary Table S2.

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. ©2013 AACR.

Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Background: Squamous cell carcinoma of the head and neck (SCCHN) is a radiation-sensitive disease. Standard frontline treatment of locally advanced SCCHN entails concurrent chemotherapy with radiation. Treatment of relapsed or metastatic SCCHN often utilizes re-irradiation to the primary tumor for local control. However, there is no standard systemic treatment for relapsed or metastatic SCCHN in combination with radiation. Many SCCHN tumors display alterations in expression of DNA repair genes. To this end, we evaluated the effect of talazoparib (BMN-673), a potent poly-ADP ribose polymerase (PARP) inhibitor, alone or in combination with radiation, in vitro and in vivo. Methods: The 50% inhibitory concentration (IC50) of talazoparib was determined in 30 SCCHN cell lines in vitro. Using clonogenic assays, the effects of 0, 10 or 100nM talazoparib, alone or in combination with a single fraction of 4Gray radiation were determined in four cell lines (two sensitive and two resistant to talazoparib). Effects on DNA damage of talazoparib, alone or in combination with radiation were also determined by flow cytometric analysis of Gamma-H2A.X. Finally, the FaDu head and neck cells were implanted onto female J/nude. After tumors were established, mice were treated with talazoparib for 5 days and a total of 20 Gray radiation, alone or in combination, and effects on body weight and tumor size were determined. Results: Among 30 SCCHN cell lines treated with talazoparib alone, 18 displayed sensitivity with 50% inhibitory concentration (IC50) of 1μM or less. In clonogenic assays, addition of radiation to BMN673 in two sensitive (UMSCC-6 and UMSCC-38) and two resistant (UMSCC-5 and UMSCC-12) cell lines resulted in synergistic cytotoxicity at doses ranging from 0-100nM BMN673. Increased gamma-H2A.X was seen when cell lines were treated with talazoparib, radiation, or the combination, compared to controls. Combining radiation with talazoparib resulted in a transient decrease in body weight in J/nude mice, but resulted in statistically significant and prolonged decrease in tumor volume. These anti-tumor effects were sustained out to 60 days post inoculation. Conclusions: The PARP inhibitor talazoparib with radiation synergistically inhibited tumor growth in vitro and in vivo in SCCHN cell lines. Further evaluation of this combination is underway. Citation Format: Deborah JL Wong, David D. Shin, Josephine Ratikan, Meenal Chalukya, Kanthinh Manivong, Leonard Post, Dörthe Schaue, Richard S. Finn, Yuqiao (Jerry) Shen, William McBride, Dennis J. Slamon. Potent anti-tumor activity of talazoparib (BMN673) in combination with radiation for squamous cell carcinoma of the head and neck. [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 1795. doi:10.1158/1538-7445.AM2015-1795

To perform candidate gene screening for posterior polymorphous corneal dystrophy (PPCD). The initial 3 genes chosen, ID1, BCL2L1, and VSX1, lie within the region on chromosome 20 to which the PPCD gene has been linked, and mutations in VSX1 have previously been identified in patients with PPCD.DNA extraction, PCR amplification, and direct sequencing of the VSX1, BCL2L1, and ID1 genes were performed in 14 affected patients (12 families) as well as in unaffected family members and healthy control subjects.No coding region mutations in the BCL2L1 or ID1 genes were identified in affected patients. In the VSX1 gene, the previously identified Gly160Asp missense change was not present in any of our 12 probands, and the Asp144Glu mutation was identified in 1 affected patient as well as 1 unaffected control individual. Additionally, 2 synonymous substitutions were identified, Ala182Ala (8 affected patients from 8 families) and Gly239Gly (1 affected patient and 1 unaffected patient from the same family). In the ID1 gene, the synonymous substitution Gly216Gly was observed in 2 affected patients (2 families) who also demonstrated a single nucleotide change in both the 5'UTR (2129TC) and 3'UTR (3267AG). Another 5'UTR change, 2177TC, was identified in 1 affected patient and his unaffected parent, both of whom also demonstrated the 2129TC and 3267AG changes.None of the 12 probands with PPCD demonstrated the previously described Gly160Asp mutation within the VSX1 gene. The Asp144Glu missense change, present in an affected patient as well as an unaffected control individual, appears to be a rare polymorphism, not a disease-causing mutation. No coding region changes were identified in the ID1 or BCL2L1 genes. Therefore, although we report a number of novel polymorphisms in the VSX1 and ID1 genes, the failure to identify any sequence variants that sort with the disease phenotype suggests that other genetic factors are involved in PPCD.

Purpose: Keratoconus is a noninflammatory, corneal thinning disorder leading to mixed myopic and irregular astigmatism and implicated as a major reason for cornea transplantations in the Western world. Genetic factors have been suggested as a cause of keratoconus. The levels of transforming growth factor β-induced (TGFBI) protein have been reported to be altered in keratoconus tissues. Mutations in this gene are responsible for causing various corneal dystrophies. Given this strong evidence of the involvement of this gene in corneal dystrophies, we investigated possible mutations within this gene in 15 probands of families with keratoconus. Methods: All patients and control individuals had complete ophthalmological examination by a corneal specialist to determine their affectation status. The entire transcript of the TGFBI gene was analyzed by direct sequencing from patient DNA. Results: We found 8 sequence variations within the gene, none of which was protein-altering changes. These changes were also observed in control individuals, and 4 are previously known polymorphisms. Conclusions: We concluded that the TGFBI gene is not responsible for causing keratoconus in these patients.

Bleparophimosis ptosis epicanthus inversus syndrome (BPES) is a rare disorder characterized by eyelid malformation and in some cases associated with premature ovarian failure. Although the familial form is autosomal dominant, many cases are also sporadic. The mutations causing this disorder were found in a winged/forkhead transcription factor gene named FOXL2. We have sequenced the mouse homolog for the FOXL2 gene and identified the Fugu rubripes (pufferfish) ortholog from the database. By alignment of the three sequences, we found an almost complete conservation of the forkhead domain in the three species. There is 95% and 61% conservation at the protein level between human–mouse and human–pufferfish, respectively. The polyalanine and polyproline tracts within the gene are absent in Fugu rubripes. An overview identifies four breaks in the conservation of the gene within these species. Using a direct sequencing approach, we performed mutation analysis from DNA of nine affected individuals from familial and sporadic cases. The mutations are distributed throughout the coding region of the FOXL2 gene. We identified five novel mutations: g.292delG (E19fsX149); g.530G>A (W98X); g.548A>G (H104R); g.652G>T (E139X); and g.1178underscore;1185del8 (A314fsX530). In addition we also identified two known mutations g.823C>T (Q196X) and g.1092underscore;1108dup17, the latter in individuals from three unrelated pedigrees. Hum Mutat 22:222–228, 2003. © 2003 Wiley-Liss, Inc.

Cone rod dystrophy 5 (CORD5) is an autosomal dominant retinal disease that primarily affects cone function. The locus has previously been mapped to human chromosome 17p12-p13 between the markers D17S926/D17S849 and D17S945/D17S804. One of our “unaffected” recombinant individual from family 1175 was subsequently found to cross through this interval. Reexamination revealed that he was in fact mildly affected. This expanded the minimum candidate region. Direct sequencing of the GUCY2D and other candidate genes within this interval was carried out on 2 American families affected with CORD5. There was an R838C missense mutation within the GUCY2D gene in one and a R838H missense mutation in another families. The previously reported mutations for CORD6 are clustered at the same position within the gene. These results indicate that both CORD5 (MIM# 600977) and CORD6 (MIM# 601777) are actually the same disease. We conclude that significant variability in expression and incomplete penetrance exists even within one family. © 2003 Wiley-Liss, Inc.

Age-related maculopathy (ARM) is a multifactorial disorder known to have a substantial genetic component. The epsilon4 allele of the apolipoprotein E gene (APOE-4) has previously been reported to have a protective effect on ARM risk, while the APOE-2 allele may increase disease risk. This study combined four independent data sets (three US and one European) of Caucasian ARM patients and controls in order to obtain better statistical power to examine the role of APOE in ARM. APOE genotype and allele frequencies were compared for 617 ARM cases and 1260 controls, adjusting for age and sex differences between the two groups via multiple logistic regression. The protective effect of the APOE-4 allele on ARM risk was confirmed (age- and sex-adjusted odds ratio (OR) for APOE-4 carriers 0.54, 95% confidence interval (CI) 0.41-0.70, p < 0.0001). The effect of APOE-4 did not differ significantly between males and females and was observed consistently for both atrophic and neovascular ARM. Evidence for an increased risk of ARM due to the APOE-2 allele was found for men, but not for women (OR for men 1.54, 95% CI 0.97-2.45; OR for women 0.74, 95% CI 0.52-1.06, p = 0.01 for interaction of sex and APOE-2 carrier status). These data confirm that the APOE-4 allele, or an allele in linkage disequilibrium with it, reduces the risk of ARM. They also suggest that the effect of the APOE-2 allele may vary by gender, and that APOE-2 may confer an increased risk only to males.

BACKGROUND: MAPK pathway dysregulation is frequently seen in cancer. For NSCLC, this may be due to driver mutations or gene amplification in EGFR, KRAS, or BRAF, though tumors without such mutations frequently display pathway hyperactivity. Aberrant EGFR signaling in SCCHN may lead to hyperactivity of the MAPK or PI3K pathways. MEK162 (ARRY-438162) is a potent, tight binding, uncompetitive MEK inhibitor currently in clinical development for treatment of solid tumors. MEK162 has an IC50 of 10nM against purified MEK1/2. We evaluated whether MEK162 would inhibit NSCLC and SCCHN cell lines and whether specific molecular alterations predicted anti-tumor activity. We also evaluated the combination of MEK162 in combination with BYL719, a potent PI3K alpha inhibitor. METHODS: The 50% inhibitory concentration (IC50) of MEK162, alone or in combination with BYL719, was determined in 40 human NSCLC and 30 SCCHN cell lines in vitro. Effects on MAPK and PI3K/AKT pathway signaling were studied by western blot analysis. Effects of MEK162 alone or in combination with BYL719 on cell cycle progression were also determined by flow cytometry. RESULTS: Using previously published cut-offs for sensitivity in cell lines, among NSCLC cell lines, 20/40 (50%) were sensitive to MEK162 (IC50 ≤ 500nM), 9/40 were intermediately sensitive (IC50 500nM-1µM), and 11/40 were resistant (IC50 ≥ 1µm). Using the same cut-offs, 80% (24/30) of SCCHN cell lines were sensitive, 2/30 were intermediately sensitive, and 4/30 were resistant to MEK162. MEK162 exposure resulted in increased cells in G0/G1 and decreased cells in S-phase. MEK162 decreased phosphorylated ERK (p-ERK) in all cell lines treated and resulted in an increase in phosphorylated AKT (p-AKT), presumably as a compensatory result from MAPK pathway inhibition. Combined treatment with MEK162 and BYL719 resulted in synergistic growth inhibition for all cell lines and decreased levels of both p-ERK and p-AKT levels at 24 hours. CONCLUSION: MEK162 potently inhibited growth of human NSCLC and SCCHN cell lines in most cell lines tested and led to synergistic inhibition when combined with BYL719. The compensatory increase in pAKT seen with MEK162 was abrogated with dual treatment with MEK162 and BYL719, demonstrating that synergy may be mediated by simultaneous blockade of MAPK and PI3K/AKT pathways. These data provide a preclinical rationale for evaluating MEK162, alone or in combination with BYL719, in metastatic NSCLC and SCCHN. Supported by 1K23CA149079, P50 CA090440, V Foundation for Cancer Research, Jonsson Comprehensive Cancer Center, Wolfen Family Lung Cancer Research Program, Stiles Program in Oncology, National Lung Cancer Partnership and One Ball Matt Memorial Golf Tournament. Citation Format: Deborah JL Wong, Edward B. Garon, Danielle D. Silveira, Naeimeh Kamranpour, Sharon Pitts, Meenal Chalukya, Habib Hamidi, Steven Dubinett, Ronald Linnartz, Richard S. Finn, Dennis J. Slamon. Potent anti-tumor activity of the MEK1/2 inhibitor MEK162 in human non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2014-743

Background and purpose: Current treatment of head and neck cancer generally involves a combination of surgery, radiation and chemotherapy. Clinical outcomes for many patients remain suboptimal, and novel therapeutic strategies are needed. Targeted inhibition of poly(ADP-Ribose) polymerase (PARP), a key DNA repair enzyme, has been actively investigated in cancer medicine. BMN 673 is a potent orally active inhibitor of PARP, currently in phase I clinical trials in solid and hematologic malignancies. We hypothesized BMN 673 might have single agent activity in a subset of head and neck cancers and could significantly augment the effects of radiation in this disease. To test this strategy, we performed preclinical studies to evaluate single agent activity of BMN 673 in a large panel of human head and neck cell lines. We also aimed to define interactions between BMN 673 and radiation in these models. Materials and methods: Human head and neck cell lines (N=32) were treated with single-agent BMN 673 for 5 days, and in vitro growth inhibition assays were performed. Further, four cell lines were studied in colony formation assays by seeding and treating them with BMN 673 at 1, 10, or 100 nM (or vehicle) for 24 hours, followed by mock or 4 Gy irradiation (300kV, 10mA). Cells were incubated for 14 or 20 days and stained with crystal violet for colony enumeration. Each experiment was performed in triplicates. Survival fraction was calculated: (number of colonies for treated cells/number of cells plated)/(number of colonies for corresponding control/number of cells plated). Results: A gradation of sensitivity was observed with BMN 673 monotherapy: IC 50 ranges from 0.1 to 10 μM. Radiation alone reduced colony forming ability of the cells by 26% in UMSCC-5 and by approximately 65% in other three cell lines (UMSCC-6, 12, and 38). These cells exhibited enhanced cytotoxicity with addition of BMN 673 to radiation in a dose-dependent manner. Addition of 100nM of BMN 673 resulted in 80% reduction in the number of surviving colonies in the relatively radio-resistant UMSCC-5 and in 88-100% reduction in other cell lines. All changes were statistically significant. Conclusions: In vitro assays demonstrate human head and neck cancer cells exhibit relative growth sensitivity to BMN 673 monotherapy. In addition, radiation-induced cytotoxicity is augmented by the addition of BMN 673 to radiation in a dose-dependent manner. Notably, one relatively radio-resistant line was rendered radio-sensitive with the addition of BMN 673 to radiation. These data support a role of PARP inhibition as monotherapy and/or in combination with radiotherapy, representing a potential novel translational therapeutic strategy in head and neck cancers that warrant clinical testing. Citation Format: David D. Shin, Josephine Ratikan, Kanthinh Manivong, Meenal Chalukya, Leonard Post, Yuqiao (Jerry) Shen, William McBride, Dörthe Schaue, Dennis J. Slamon, Richard S. Finn. The poly(ADP-ribose) polymerase inhibitor BMN 673 has single agent activity and augments cytotoxicity of radiation in human head and neck tumor cell line in vitro: a novel strategy for radiosensitization in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2013-1595

Background: Cell cycle dysregulation is a common molecular finding in ovarian cancer and the cyclin-D kinases (CDK) represent an attractive target in this pathway. PD 0332991 (Pfizer Inc.) is an orally active potent and highly selective inhibitor of CDK 4 and CDK6 kinases with the ability to block pRb phosphorylation at serine 780 and 795 in the low nanomolar range. To identify predictors of response to PD 0332991, we determined the in vitro sensitivity to PD 0332991 in a large panel of molecularly characterized ovarian cancer cell lines, and then used baseline expression profiles and Western blot analyses to identify biomarkers associated with response to PD 0332991. These biomarkers were then validated in a large cohort of 324 ovarian cancer patients by immunohistochemistry. Methods: 40 human ovarian cancer cell lines were treated with PD 0332991 using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate the IC50 across the panel. These data were then analyzed against baseline gene expression data to identify genes associated with sensitivity to PD 0332991. Western blot analysis was performed to validate these markers and to study the effects of PD 0332991 on pRb phosphorylation. Cell cycle analysis was performed using flow-cytometry. Combination studies were performed to analyze the interaction between PD 0332991 and paclitaxel or carboplatin chemotherapy. Tissue microarrays were constructed from 324 ovarian cancer patients and IHC was performed for p16 and Rb expression. Long-term clinical follow up was available for all patients. Results: Concentration-dependent anti-proliferative effects of PD 0332991were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC50s (IC50 range: 0.02 to >20 μmol/L). Cell lines with high Rb expression and low p16 expression by gene expression array and Western blot analyses were most sensitive to inhibition by PD 0332991. Cell lines treated with PD 0332991 showed clear G0/G1 arrest and a decrease in S-phase fraction in sensitive but not in resistant cell lines. In addition, Western blot showed that pRb phosphorylation is blocked in a concentration and time dependent manner. IHC for Rb and p16 allows the identification of ovarian cancer patients most likely to benefit from CDK4/6 inhibition. Outcome analyses are ongoing. Conclusion: PD 0332991 shows promising biologic activity in ovarian cancer cells. Assessment of Rb and p16 expression may help select patients who benefit from therapeutic strategies targeting CDK4/6 in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 25.

Patients with chronic myeloid leukemia (CML) are experiencing prolonged survival due to successful therapy with tyrosine kinase inhibitors. However, some CML patients who have achieved longstanding remissions with these agents harbor clonal cytogenetic abnormalities in their Philadelphia chromosome negative (Ph-) bone marrow cells. Because CML patients in remission often have peripheral blood count abnormalities, including cytopenias, we investigated whether these patients may have developed myelodysplastic syndrome (MDS) within the Ph- cell population. Bone marrow samples from 26 CML patients who had achieved a major cytogenetic remission (MCyR) with tyrosine kinase inhibitor therapy between 2 and 15 years after diagnosis were evaluated; 6 patients had advanced disease prior to their last therapy, 20 were in chronic phase. At the time of evaluation, 2 of the patients were receiving imatinib, 23 dasatinib, and 1 PHA739358. At least one peripheral blood lineage was abnormal in 21 patients, of whom 7 had pancytopenia. Routine metaphase cytogenetics (MC) revealed a persistent clonal chromosomal abnormality in 10% of the Ph- metaphases in 5 patients (+8 in 2, −7 in 2, and 20q- in 1). We hypothesized that clonal hematopoiesis might exist in additional patients and applied single nucleotide array (SNP-A) based karyotyping and X-linked human androgen receptor (HUMARA) clonality assay to further delineate the nature of the hematopoietic defect in these patients. HUMARA was performed on bone marrow samples and germ-line DNA from peripheral blood T lymphocytes of the female patients. Clonality, as assessed by skewing of X-chromosome inactivation in bone marrow cells compared to germline control cells, could not be demonstrated in the12 female patients. SNP-A karyptyping using 250K Affymetrix SNP array confirmed the known cytogenetic abnormalities. Several microdeletions were found, but comparison with purified T lymphocytes demonstrated that these “lesions” represented germ line-encoded copy number variants. However, SNP-A karyotyping revealed the presence of uniparental disomy (UPD) involving chromosome 17(p12-pter) in bone marrow, but not germ line cells, from one male patient with normal karyotype by routine MC. In the context of secondary AML, del17p or UPD17 have been observed always in the presence of del7/q and 5q and were associated with poor prognosis. However, in our patient UPD17 occurred as a sole defect. Because in our studies in AML, UPD of chromosome 17p was found in association with p53 mutations, genomic sequencing of this gene was performed. A 5 bp deletion destroying the splice acceptor region of exon 6 was identified in bone marrow cells from this patient. Alternative splicing leading to loss of exon 6 was predicted to result in a frame shift and premature introduction of a stop codon. These methods revealed clonal hematopoiesis in the Ph- bone marrow cells of 6/26 patients with longstanding CML in remission from tyrosine kinase inhibitors and persistent peripheral blood abnormalities. The approaches used here probably underestimate the frequency of this condition, as oligoclonal populations may be present in numbers below the limit of assay sensitivity. The Ph- clonal bone marrow populations have cytogenetic and molecular features in common with MDS. After a median follow up of two years, one patient with monosomy 7 developed acute myeloid leukemia, but longer follow up will be required to determine the natural history of the Ph- clonal disorders.

Background. The T315I BCR-ABL mutation confers high level resistance to clinically available ABL kinase inhibitors (i.e., imatinib, nilotinib, dasatinib, bosutinib, INNO-406). PHA-739358 is an aurora kinase inhibitor that selectively inhibits the ATP site of Aurora-A, Aurora-B and Aurora-C kinases, and binds with high affinity to both wild type Abl and Abl/T315I in vitro. A multicenter phase II study of PHA-739358 is being conducted in patients with chronic myeloid leukemia (CML) relapsing on imatinib or other c-ABL therapy. Methods. Seven consenting CML patients (1 in chronic phase, 1 in accelerated phase, 5 in blast phase) were initially entered in this trial and received PHA-739358 dosed at 250 or 330 mg/m2/day administered by a once-weekly 6-hour infusion for 3 consecutive weeks, every 4 weeks. Six out of seven patients had the BCR-ABL T315I mutation. Pharmacokinetic and pharmacodynamic (PD) samples were collected and analyzed during cycle 1. The PD analyses evaluated BCR-ABL inhibition (phospho-CRKL) and Aurora kinase inhibition (phospho-histone H3). Results. Two patients with T315I mutated BCR-ABL achieved a complete hematologic response (CHR) to PHA-739358. One of these patients treated in accelerated phase achieved a complete cytogenetic response (CCyR) and a complete molecular response after 3 months on the 330 mg/m2 dose level. The CCyR is ongoing after 6 months of treatment. The second patient who achieved a CHR initiated therapy in chronic phase. A minor cytogenetic response was achieved after 3 cycles of therapy at the 330 mg/m2, and a minimal cytogenetic response persists after nine months of treatment. Uncomplicated grade 4 neutropenia and an infusion-related reaction requiring acetaminophen, benadryl and hydrocortisone premedication was observed in one patient. No other grade 3/4 non-hematologic toxicity was observed. Patients with blast crisis did not have CHRs at the dose and schedule employed, although suppression of peripheral blood blasts was evident. PHA-739358 pharmacokinetics were in good agreement with those observed in other phase I - II studies. Average exposure, AUC0-t(168h), observed at 330 mg/m2/day was about 45 uM/h with Cmax values around 4 – 6 uM/h. Pharmacodynamic analyses demonstrated treatment-associated decreases of CRKL phosphorylation in 6 out of 7 patients, including both responders. Modulation of histone H3 phosphorylation was observed in 3 of 5 evaluable patients. Conclusions. Objective clinical responses to PHA-739358 have been observed in two CML patients with T315I mutations of BCR-ABL, with an acceptable tolerability and safety profile. Additional doses and schedules of PHA-739358 are being investigated in patients with advanced phase CML.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharo-phimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype-phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and III by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.