Key Points TORK/DNA-PK inhibition induces cytotoxicity and blocks signaling pathways important for CLL survival, proliferation, and drug resistance. Preliminary clinical effects of TORK/DNA-PK inhibition show 7 of 8 CLL patients with decreased lymphadenopathy.

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 bloodderived 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 crosslinking of GA101 and involved a lysosomedependent 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. (Blood. 2011;118(19): 5178-5188) IntroductionAlthough treatment results for chronic lymphocytic leukemia (CLL) have improved considerably over the last decade, a curative drug regimen is still lacking. Similar to other B-cell malignancies such as follicular lymphoma and multiple myeloma, in CLL, the interaction of the malignant cells with their microenvironment in the lymph nodes, spleen, and possibly BM has been shown to play an important role in the biology of the disease. 1 We and others have previously shown that in vitro CD40 stimulation of CLL cells can to a certain extent mimic the lymph node environment and results in the induction of resistance of the CLL cells to cytotoxic drugs such as fludarabine, chlorambucil, bortezomib, and roscovitine. [2][3][4][5][6] These microenvironmental niches might be an important localization of minimal residual disease and form the basis for the relapses characterizing this disease. 1,[3][4][5]7,8 Moreover, after sequential treatments, selection of p53 dysfunctional clones occurs in up to 50% of patients, 9,10 which also results in chemoresistance. Therefore, there is a need for new treatments that circumvent microenvironmental chemoresistance and act independently of p53, possibly including anti-CD20 mAb-containing regimens. 11,12 However, sensitivity of CLL cells to anti-CD20 mAbs in vitro is low and monotherapy with conventional doses of the type I anti-CD20 mAb rituximab has only limited efficacy in CLL. Because of rituximab resistance or unresponsiveness, more potent anti-CD20 mAbs are currently being sought. Two types of anti-CD20 mAbs have been described. A prime difference is that, in contrast to type I anti-CD20 mAbs, type II mAbs are unable to translocate CD20 into lipid rafts or to evoke Ca 2ϩ flux. [13][14][15][16] Ofatumumab, a second-generation type I anti-CD20 mAb seems promising for the treatment of CLL, 17 although large amounts seem to be required. GA101 is a novel glycoengineered type II anti-CD20 mAb. Compared with rituximab, GA101 has enhanced direct cell death-inducing capacity and improved Ab-depende...

In vitro CD40-stimulated chronic lymphocytic leukemia (CLL) cells are resistant to cytotoxic drugs. In sharp contrast, we here show that CD40 stimulation sensitizes CLL cells to rituximabmediated cell death. This increased sensitivity is specific for anti-CD20 treatment. Rituximab-mediated death in CD40-stimulated CLL cells shows rapid kinetics (within hours), and is caspase and p53 independent, but depends on extracellular Ca 2 þ and reactive oxygen species (ROS) production. By increasing basal ROS production, CD40 stimulation sensitizes CLL cells to rituximab-mediated death. Our findings provide a rationale for combination treatment of CLL with cytotoxic drugs and anti-CD20 monoclonal antibodies.

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.