Philip Sutera

Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737–Mediated Cell Death in Malignant Human Glioma Cell Lines

The prognosis for malignant glioma, the most common brain tumor, is still poor, underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell-cycle control, there is a strong rationale for examining the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact, deleted, or mutated p53 or phosphatase and tensin homolog on chromosome 10; intact or deleted or p14ARF or wild-type or amplified epidermal growth factor receptor. Dinaciclib inhibited cell proliferation and induced cell-cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72-hour 3-[4,5-dimethylthiazol- 2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H, tetrazolium (MTS) assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed, but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth-signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitors ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide) or ABT-737 (4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential; the release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor; phosphatidylserine exposure on the plasma membrane surface and activation of caspases and poly ADP-ribose polymerase. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1. These observations may have important clinical implications for the design of experimental treatment protocols for malignant human glioma.

Survivin inhibitor YM155 induces mitochondrial dysfunction, autophagy, DNA damage and apoptosis in Bcl‐xL silenced glioma cell lines

Because the anti‐apoptotic protein Bcl‐xL is overexpressed in glioma, one might expect that inhibiting or silencing this gene would promote tumor cell killing. However, our studies have shown that this approach has limited independent activity, but may tip the balance in favor of apoptosis induction in response to other therapeutic interventions. To address this issue, we performed a pharmacological screen using a panel of signaling inhibitors and chemotherapeutic agents in Bcl‐xL silenced cells. Although limited apoptosis induction was observed with a series of inhibitors for receptor tyrosine kinases, PKC inhibitors, Src family members, JAK/STAT, histone deacetylase, the PI3K/Akt/mTOR pathway, MAP kinase, CDK, heat shock proteins, proteasomal processing, and various conventional chemotherapeutic agents, we observed a dramatic potentiation of apoptosis in Bcl‐xL silenced cells with the survivin inhibitor, YM155. Treatment with YM155 increased the release of cytochrome c, smac/DIABLO and apoptosis inducing‐factor, and promoted loss of mitochondrial membrane potential, activation of Bax, recruitment of LC3‐II to the autophagosomes and apoptosis in Bcl‐xL silenced cells. We also found an additional mechanism for the augmentation of apoptosis due to abrogation of DNA double‐strand break repair mediated by Rad51 repression and enhanced accumulation of γH2AX. In summary, our observations may provide a new insight into the link between Bcl‐xL and survivin inhibition for the development of novel therapies for glioma.

One- vs. Three-Fraction Pancreatic Stereotactic Body Radiation Therapy for Pancreatic Carcinoma: Single Institution Retrospective Review

Background/introduction Early reports of stereotactic body radiation therapy (SBRT) for pancreatic ductal adenocarcinoma (PDAC) used single fraction, but eventually shifted to multifraction regimens. We conducted a single institution review of our patients treated with single- or multifraction SBRT to determine whether any outcome differences existed. Methods and materials Patients treated with SBRT in any setting for PDAC at our facility were included, from 2004 to 2014. Overall survival (OS), local control (LC), regional control (RC), distant metastasis (DM), and late grade 3 or greater radiation toxicities from the time of SBRT were calculated using Kaplan–Meier estimation to either the date of last follow-up/death or local/regional/distant failure. Results We identified 289 patients (291 lesions) with pathologically confirmed PDAC. Median age was 69 (range, 33–90) years. Median gross tumor volume was 12.3 (8.6–21.3) cm3 and planning target volume 17.9 (12–27) cm3. Single fraction was used in 90 (30.9%) and multifraction in 201 (69.1%) lesions. At a median follow-up of 17.3 months (IQR 10.1–29.3 months), the median survival for the entire cohort 17.8 months with a 2-year OS of 35.3%. Univariate analysis showed multifraction schemes to have a higher 2-year OS 30.5% vs. 37.5% (p = 0.019), it did not hold significance on MVA. Multifractionation schemes were found to have a higher LC on MVA (HR = 0.53, 95% CI, 0.33–0.85, p = 0.009). At 2 years, late grade 3+ toxicity was 2.5%. Post-SBRT CA19-9 was found on MVA to be a prognostic factor for OS (HR = 1.01, 95% CI, 1.01–1.01, p = 0.009), RC (HR = 1.01, 95% CI 1.01–1.01, p = 0.02), and DM (HR = 1.01, 95% CI, 1.01–1.01, p = 0.001). Conclusion Our single institution retrospective review is the largest to date comparing single and multifraction SBRT and the first to show multifraction regimen SBRT to have a higher LC than single fractionation. Additionally, we show low rates of severe late toxicity with SBRT.

Results of a prospective phase 2 clinical trial of induction gemcitabine/capecitabine followed by stereotactic ablative radiation therapy in borderline resectable or locally advanced pancreatic adenocarcinoma

PURPOSE Stereotactic ablative radiation therapys (SABRs) great conformity and short duration has become an attractive treatment modality. We report a phase 2 clinical trial to evaluate efficacy and safety of induction chemotherapy (ICT) followed by SABR in patient with borderline resectable (BR) and locally advanced (LA) pancreatic ductal adenocarcinoma (PDAC). METHODS AND MATERIALS Patients with biopsy-proven BR or LA PDAC were treated with four 21-day cycles of intravenous gemcitabine and oral capecitabine. Patients were restaged within 4 weeks after ICT by computed tomography and treated by 3-fraction SABR if no metastasis or progressive disease was identified. Patients were restaged 4 weeks following SABR to determine resectability. Tumor response was assessed with carbohydrate antigen 19-9. RESULTS Thirty-five patients (19 BR/16 LA) were enrolled. The median age was 71.8 years (range, 50.6-81.1). ICT was completed in 91.4% (n = 32) of patients. All patients who completed ICT completed SABR. Of those 32 patients, 34.3% (n = 12: 10 BR, 2 LA) underwent pancreaticoduodenectomy and 11 of 12 (91.7%) received R0 resection. Median overall survival was 18.8, 28.3, and 14.3 months for the entire cohort, BR, and LA, respectively. The 2-year local progression-free survival (LPFS) was 44.9%, 40%, and 52% for the entire cohort, BR, and LA, respectively. For BR patients, multivariate analysis showed surgery was associated with better overall survival and LPFS. One-year LPFS for patients with surgery was 80% and 44% without surgery. Within the 15.4-month follow-up, no grade 3+ toxicity from SABR was observed. No significant quality of life change was observed before and after ICT, SABR, or surgery for BR or LA patients. CONCLUSIONS This is the first prospective phase 2 study to investigate the feasibility and efficacy of a 12-week gemcitabine/capecitabine ICT followed by SABR for BR or LA PDAC. The results suggest excellent tolerability, high R0 resection rates, and acceptable posttreatment complications.

Mitochondrial dysfunction RAD51, and Ku80 proteolysis promote apoptotic effects of Dinaciclib in Bcl-xL silenced cells

In the present study, we investigated the effect of CDK inhibitors (ribociclib, palbociclib, seliciclib, AZD5438, and dinaciclib) on malignant human glioma cells for cell viability, apoptosis, oxidative stress, and mitochondrial function using various assays. None of the CDK inhibitors induced cell death at a clinically relevant concentration. However, low nanomolar concentrations of dinaciclib showed higher cytotoxic activity against Bcl‐xL silenced cells in a time‐ and concentration‐dependent manner. This effect was not seen with other CDK inhibitors. The apoptosis‐inducing capability of dinaciclib in Bcl‐xL silenced cells was evidenced by cell shrinkage, mitochondrial dysfunction, DNA damage, and increased phosphatidylserine externalization. Dinaciclib was found to disrupt mitochondrial membrane potential, resulting in the release of cytochrome c, AIF, and smac/DIABLO into the cytoplasm. This was accompanied by the downregulation of cyclin‐D1, D3, and total Rb. Dinaciclib caused cell cycle arrest in a time‐ and concentration‐dependent manner and with accumulation of cells in the sub‐G1 phase. Our results also revealed that dinaciclib, but not ribociclib or palbociclib or seliciclib or AZD5438 induced intrinsic apoptosis via upregulation of the levels of pro‐apoptotic proteins (Bax and Bak), resulting in the activation of caspases and cleavage of PARP. We also found an additional mechanism for the dinaciclib‐induced augmentation of apoptosis due to abrogation RAD51‐cyclin D1 interaction, specifically proteolysis of the DNA repair proteins RAD51 and Ku80. Our results suggest that successfully interfering with Bcl‐xL function may restore sensitivity to dinaciclib and could hold the promise for an effective combination therapeutic strategy.

Prognostic Factors for Elderly Patients Treated With Stereotactic Body Radiation Therapy for Pancreatic Adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) commonly presents later in life with a median age at diagnosis of 70 years. Unfortunately, elderly patients are significantly underrepresented in clinical trials. Stereotactic body radiation therapy (SBRT) is a promising treatment modality in this population as it has demonstrated excellent local control with minimal toxicity. We aimed to determine prognostic factors associated with outcomes in elderly patients treated with SBRT. Materials and Methods: Elderly patients older than 70 treated with SBRT for PDAC at our institution, from 2004 to 2014 were included. Our primary endpoints included overall survival (OS) and local-progression-free survival (LPFS). Secondary endpoints included regional-progression-free survival (RPFS), distant-progression-free-survival (DPFS) and radiation toxicity. Endpoints were analyzed with the Kaplan-Meier method. The association of these survival endpoints with risk factors was studied with Cox proportional hazards models. Results: We identified 145 patients with 146 lesions of pancreatic adenocarcinoma with a median age at diagnosis of 79 (range, 70.1–90.3). SBRT was delivered to a median dose of 36 Gy (IQR 24–36). Surgical resection was performed on 33.8% of the total patients. Median follow-up was 12.3 months (IQR 6.0–23.3 months) and the median survival for the entire cohort 14.0 months with a 2-year OS of 27%. Multivariate analysis (MVA) demonstrated surgery [p ≤ 0.0001, HR 0.29 (95% CI, 0.16–0.51)] and post-SBRT CA19-9 [p = 0.009, HR 1.0004 (95% CI, 1.0002–1.0005)] significantly associated with overall survival. Recurrent lesions [p = 0.0069, HR 5.1 (95% CI, 1.56–16.64)] and post-SBRT CA19-9 levels [p = 0.0107, HR 1.0005 (95% CI, 1.0001–1.0008)] were significantly associated with local control on MVA. For the entire cohort, 4.1% experienced acute grade 2+ toxicity, and 2% experienced late grade 2+ toxicity at 2 years. Conclusion: This review demonstrates prognostic factors in elderly patients with PDAC treated with SBRT. We identified surgical resection and post-SBRT CA 19-9 as predictive of overall survival in this population. Additionally, we show low acute and late toxicity following SBRT in elderly patients.

Stereotactic Body Radiation Therapy for Locally Progressive and Recurrent Pancreatic Cancer after Prior Radiation

Introduction Pancreatic adenocarcinoma is an aggressive malignancy that has consistently demonstrated poor outcomes despite aggressive treatments. Despite multimodal treatment, local disease progression and local recurrence are common. Management of recurrent or progressive pancreatic carcinomas proves a further challenge. In patients previously treated with radiation therapy, stereotactic body radiation therapy (SBRT) is a promising modality capable of delivering high dose to the tumor while limiting dose to critical structures. We aimed to determine the feasibility and tolerability of SBRT for recurrent or local pancreatic cancer in patients previously treated with external beam radiation therapy (EBRT). Materials and methods Patients treated with EBRT who developed recurrent or local pancreatic ductal adenocarcinoma treated with SBRT reirradiation at our institution, from 2004 to 2014 were reviewed. Our primary endpoints included overall survival (OS), local control, regional control, and late grade 3+ radiation toxicity. Endpoints were analyzed with the Kaplan–Meier method. The association of these survival endpoints with risk factors was studied with univariate Cox proportional hazards models. Results We identified 38 patients with recurrent/progressive pancreatic cancer treated with SBRT following prior radiation therapy. Prior radiation was delivered to a median dose of 50.4 Gy in 28 fractions. SBRT was delivered to a median dose of 24.5 Gy in 1–3 fractions. Surgical resection was performed on 55.3% of all patients. Within a median follow-up of 24.4 months (inter-quartile range, 14.9–32.7 months), the median OS from diagnosis for the entire cohort was 26.6 months (95% CI: 20.3–29.8) with 2-year OS of 53.0%. Median survival from SBRT was 9.7 months (95% CI, 5.5–13.8). The 2-year freedom from local progression and regional progression was 58 and 82%, respectively. For the entire cohort, 18.4 and 10.5% experienced late grade 2+ and grade 3+ toxicity, respectively. Conclusion This single institution retrospective review identified SBRT reirradiation to be a feasible and tolerable treatment strategy for patients with previous locally progressive or recurrent pancreatic adenocarcinoma.

Stereotactic Ablative Radiotherapy for Unresectable Colorectal Oligometastases

Purpose Patients with oligometastatic colorectal cancer have demonstrated excellent clinical outcomes with surgical resection of hepatic and pulmonary metastases. Stereotactic ablative radiation therapy (SABR) has emerged as an alternative local therapy for nonsurgical candidates. Herein, we report the oncologic and patient-reported quality-of-life (PR-QoL) outcomes for a subset of patients with oligometastatic colorectal cancer who were treated in a prospective phase 2 multicenter clinical trial. Methods and materials Patients with a pathologically proven diagnosis of oligometastatic colorectal cancer were enrolled as part of a prospective study. SABR dose and fractionation schedules were dependent on the lesion location and size. Patient follow-up occurred 6 weeks after completion of SABR and at 3-month intervals for the following 3 years. Patients received the Functional Assessment of Cancer Therapy-General questionnaire at baseline and at each follow-up visit to assess PR-QoL. The total Functional Assessment of Cancer Therapy-General questionnaire scores were compared with those from baseline using the Wilcoxon signed rank test. Overall survival, local progression-free survival (PFS), and distant PFS were calculated using the Kaplan-Meier estimation to the date of the last follow-up visit/death or local/distant failure. Results A total of 31 patients with oligometastatic colorectal cancer with 1 (71.0%), 2 (16.1%), 3 (3.2%), 4 (3.2%), or 5 (6.5%) metastatic lesions were identified. After a median follow-up time of 50.1 months, the median OS from the time of completion of the SABR was 53.9 months (95% confidence interval, 23.2-84.6), and the 5-year OS, local PFS, and distant PFS were 45%, 83%, and 27%, respectively. Acute grade 2+ toxicity was 9.7% (pain, nausea, fatigue) and late grade 3+ toxicity (small bowel obstruction) was 3.2% with no significant change in PR-QoL in the year after SABR. Conclusions This subset analysis of a prospective phase 2 study demonstrates that SABR is a safe and effective treatment option for patients with unresectable oligometastic colorectal cancer. In addition, SABR of oligometastatic disease preserves PR-QoL.

Abstract 3565: Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines

The prognosis of malignant glioma, the most common brain tumor, is still poor, thus underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell cycle control, there is a strong rationale to examine the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact or deleted or mutated p53 or PTEN or p14ARF, or EGFR amplification status. Dinaciclib was found to inhibit cell proliferation and induce cell cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72 h MTS assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitor ABT-263 or ABT-737 with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c, smac/DIABLO and apoptosis-inducing factor (AIF), phosphatidylserine exposure on the plasma membrane surface and activation of caspases and PARP. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1. Citation Format: Esther P. Jane, Daniel Premkumar, Jonathon Cavaleri, Thatchana Rajasekar, Philip Sutera, Ian Pollack. Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3565.