Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia.

Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity however to induce cell death as monotherapy and are unlikely to eradicate the disease. Acquired resistance to therapy in CLL is often caused by mutations in the response network being targeted, both for DNA damage or BCR signaling pathways. Thus, drugs with dual targeting capacity could offer improved therapeutic value. Here, the potency of CC-115, a novel inhibitor of mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK), was evaluated in primary CLL cells in vitro and in CLL patients. Combined TORK and DNA-PK inhibition in vitro resulted in caspase-dependent cell killing irrespective of p53, ATM, NOTCH1, or SF3B1 status. Proliferation induced by CD40(+) interleukin-21 stimulation was completely blocked by CC-115, and CD40-mediated resistance to fludarabine and venetoclax could be reverted by CC-115. BCR-mediated signaling was inhibited by CC-115 and also in CLL samples obtained from patients with acquired resistance to idelalisib treatment. Clinical efficacy of CC-115 was demonstrated in 8 patients with relapsed/refractory CLL/small lymphocytic lymphoma harboring ATM deletions/mutations; all but 1 patient had a decrease in lymphadenopathy, resulting in 1 IWCLL partial response (PR) and 3 PRs with lymphocytosis. In conclusion, these preclinical results, along with early promising clinical activity, suggest that CC-115 may be developed further for treatment of CLL. The trial was registered at www.clinicaltrials.gov as #NCT01353625.

Radiosensitization by Hyperthermia Critically Depends on the Time Interval.

PURPOSE: Hyperthermia is a potent sensitizer of radiation therapy that improves both tumor control and survival in women with locally advanced cervical cancer (LACC). The optimal sequence and interval between hyperthermia and radiation therapy are still under debate. METHODS AND MATERIALS: We investigated the interval and sequence in vitro in cervical cancer cell lines, patient-derived organoids, and SiHa cervical cancer hind leg xenografts in athymic nude mice and compared the results with retrospective results from 58 women with LACC treated with thermoradiotherapy. RESULTS: All 3 approaches confirmed that shortening the interval between hyperthermia and radiation therapy enhanced hyperthermic radiosensitization by 2 to 8 times more DNA double-strand breaks and apoptosis and 10 to 100 times lower cell survival, delayed tumor growth in mice, and increased the 5-year survival rate of women with LACC from 22% (interval ≥80 minutes) to 54% (interval <80 minutes). In vitro and in vivo results showed that the sequence of hyperthermia and radiation therapy did not affect the outcome. CONCLUSIONS: Shortening the interval between hyperthermia and radiation therapy significantly improves treatment outcomes. The sequence of hyperthermia and radiation therapy (before or after) does not seem to matter.

CD40 stimulation sensitizes CLL cells to lysosomal cell death induction by type II anti-CD20 mAb GA101.

Sensitivity of chronic lymphocytic leukemia (CLL) cells to anti-CD20 mAbs is low and, therefore, the efficacy of monotherapy with current anti-CD20 mAbs is limited. At present, it is not known whether sensitivity of CLL cells to CD20 mAbs is modulated by microenvironmental stimuli. We have shown previously that in vitro CD40 stimulation of peripheral blood-derived CLL cells results in resistance to cytotoxic drugs. In the present study, we show that, in contrast, CD40 stimulation sensitizes CLL cells to the recently described novel type II anti-CD20 mAb GA101. Cell death occurred without cross-linking of GA101 and involved a lysosome-dependent mechanism. Combining GA101 with various cytotoxic drugs resulted in additive cell death, not only in CD40-stimulated CLL cells, but also in p53-dysfunctional CLL cells. Our findings indicate that GA101 has efficacy against chemoresistant CLL, and provide a rationale for combining cytotoxic drugs with anti-CD20 mAbs.

Elevated temperatures and longer durations improve the efficacy of oxaliplatin- and mitomycin C-based hyperthermic intraperitoneal chemotherapy in a confirmed rat model for peritoneal metastasis of colorectal cancer origin.

Introduction: In patients with limited peritoneal metastasis (PM) originating from colorectal cancer, cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a potentially curative treatment option. This combined treatment modality using HIPEC with mitomycin C (MMC) for 90 minutes proved to be superior to systemic chemotherapy alone, but no benefit of adding HIPEC to CRS alone was shown using oxaliplatin-based HIPEC during 30 minutes. We investigated the impact of treatment temperature and duration as relevant HIPEC parameters for these two chemotherapeutic agents in representative preclinical models. The temperature- and duration- dependent efficacy for both oxaliplatin and MMC was evaluated in an in vitro setting and in a representative animal model. Methods: In 130 WAG/Rij rats, PM were established through i.p. injections of rat CC-531 colon carcinoma cells with a signature similar to the dominant treatment-resistant CMS4 type human colorectal PM. Tumor growth was monitored twice per week using ultrasound, and HIPEC was applied when most tumors were 4-6 mm. A semi-open four-inflow HIPEC setup was used to circulate oxaliplatin or MMC through the peritoneum for 30, 60 or 90 minutes with inflow temperatures of 38°C or 42°C to achieve temperatures in the peritoneum of 37°C or 41°C. Tumors, healthy tissue and blood were collected directly or 48 hours after treatment to assess the platinum uptake, level of apoptosis and proliferation and to determine the healthy tissue toxicity. Results: In vitro results show a temperature- and duration- dependent efficacy for both oxaliplatin and MMC in both CC-531 cells and organoids. Temperature distribution throughout the peritoneum of the rats was stable with normothermic and hyperthermic average temperatures in the peritoneum ranging from 36.95-37.63°C and 40.51-41.37°C, respectively. Treatments resulted in minimal body weight decrease (<10%) and only 7/130 rats did not reach the endpoint of 48 hours after treatment. Conclusions: Both elevated temperatures and longer treatment duration resulted in a higher platinum uptake, significantly increased apoptosis and lower proliferation in PM tumor lesions, without enhanced normal tissue toxicity. Our results demonstrated that oxaliplatin- and MMC-based HIPEC procedures are both temperature- and duration-dependent in an in vivo tumor model.

PARP1-Inhibition Sensitizes Cervical Cancer Cell Lines for Chemoradiation and Thermoradiation.

Radiotherapy plus cisplatin (chemoradiation) is standard treatment for women with locoregionally advanced cervical cancer. Both radiotherapy and cisplatin induce DNA single and double-strand breaks (SSBs and DSBs). These double-strand breaks can be repaired via two major DNA repair pathways: Classical Non-Homologous End-Joining (cNHEJ) and Homologous Recombination. Besides inducing DNA breaks, cisplatin also disrupts the cNHEJ pathway. Patients contra-indicated for cisplatin are treated with radiotherapy plus hyperthermia (thermoradiation). Hyperthermia inhibits the HR pathway. The aim of our study is to enhance chemoradiation or thermoradiation by adding PARP1-inhibition, which disrupts both the SSB repair and the Alternative NHEJ DSB repair pathway. This was studied in cervical cancer cell lines (SiHa, HeLa, C33A and CaSki) treated with hyperthermia (42 °C) ± ionizing radiation (2-6 Gy) ± cisplatin (0.3-0.5 µM) ± PARP1-inhibitor (olaparib, 4.0-5.0 µM). Clonogenic assays were performed to measure cell reproductive death. DSBs were analyzed by γ-H2AX staining and cell death by live cell imaging. Both chemoradiation and thermoradiation resulted in lower survival fractions and increased unrepaired DSBs when combined with a PARP1-inhibitor. A quadruple modality, including ionizing radiation, hyperthermia, cisplatin and PARP1-i, was not more effective than either triple modality. However, both chemoradiation and thermoradiation benefit significantly from additional treatment with PARP1-i.

Pre-Operative Decitabine in Colon Cancer Patients: Analyses on WNT Target Methylation and Expression.

DNA hypermethylation is common in colon cancer. Previously, we have shown that methylation of WNT target genes predicts poor prognosis in stage II colon cancer. The primary objective of this study was to assess whether pre-operative treatment with decitabine can decrease methylation and increase the expression of WNT target genes APCDD1, AXIN2 and DKK1 in colon cancer patients. A clinical study was conducted, investigating these potential effects of decitabine in colon cancer patients (DECO). Patients were treated two times with 25 mg/m2 decitabine before surgery. Methylation and expression of LINE1 and WNT target genes (primary outcome) and expression of endogenous retroviral genes (secondary outcome) were analysed in pre- and post-treatment tumour samples using pyrosequencing and rt-PCR. Ten patients were treated with decitabine and eighteen patients were used as controls. Decitabine treatment only marginally decreased LINE1 methylation. More importantly, no differences in methylation or expression of WNT target or endogenous retroviral genes were observed. Due to the lack of an effect on primary and secondary outcomes, the study was prematurely closed. In conclusion, pre-operative treatment with decitabine is safe, but with the current dosing, the primary objective, increased WNT target gene expression, cannot be achieved.

The Temperature-Dependent Effectiveness of Platinum-Based Drugs Mitomycin-C and 5-FU during Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in Colorectal Cancer Cell Lines.

Cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment with curative intent for peritoneal metastasis of colorectal cancer (CRC). Currently, there is no standardized HIPEC protocol: choice of drug, perfusate temperature, and duration of treatment vary per institute. We investigated the temperature-dependent effectiveness of drugs often used in HIPEC. METHODS: The effect of temperature on drug uptake, DNA damage, apoptosis, cell cycle distribution, and cell growth were assessed using the temperature-dependent IC50 and Thermal Enhancement Ratio (TER) values of the chemotherapeutic drugs cisplatin, oxaliplatin, carboplatin, mitomycin-C (MMC), and 5-fluorouracil (5-FU) on 2D and 3D CRC cell cultures at clinically relevant hyperthermic conditions (38-43 °C/60 min). RESULTS: Hyperthermia alone decreased cell viability and clonogenicity of all cell lines. Treatment with platinum-based drugs and MMC resulted in G2-arrest. Platinum-based drugs display a temperature-dependent synergy with heat, with increased drug uptake, DNA damage, and apoptosis at elevated temperatures. Apoptotic levels increased after treatment with MMC or 5-FU, without a synergy with heat. CONCLUSION: Our in vitro results demonstrate that a 60-min exposure of platinum-based drugs and MMC are effective in treating 2D and 3D CRC cell cultures, where platinum-based drugs require hyperthermia (>41 °C) to augment effectivity, suggesting that they are, in principle, suitable for HIPEC.