The near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

Enhanced expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) protein is frequent in cancer. Targeting of BCL-2 with the specific inhibitor ABT-199 (Venetoclax) has significant clinical activity in malignant diseases such as chronic lymphocytic leukemia and multiple myeloma. The small molecule drug ABT-199 mimics the pro-apoptotic BCL-2 homology domain 3 of BH3-only proteins and blocks the hydrophobic BC-groove in BCL-2. We have previously shown that ABT-199 synergizes with the proteasome inhibitor (PI) bortezomib in soft tissue sarcoma derived cells and cell lines to induce apoptosis. Synergistic apoptosis induction relies on the pore-forming effector BAX and expression of the pro-apoptotic BH3-only protein NOXA. Bortezomib augments expression of NOXA by blocking its proteasomal degradation. Interestingly, shown here for the first time, expression of NOXA is strongly enhanced by ABT-199 induced integrated stress response (ISR). ISR transcription factors ATF3 & ATF4 mediate transactivation of the BH3-only protein NOXA which specifically inhibits the anti-apoptotic MCL-1. Thus, NOXA potentiates the efficacy of the BCL-2 inhibitor ABT-199 by simultaneous inhibition of MCL-1. Hence, ABT-199 has a double impact by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivated NOXA. By preventing degradation of NOXA PIs synergize with ABT-199. Synergism of ABT-199 and PIs therefore occurs on several, previously unexpected levels. This finding should prompt clinical evaluation of combinatorial regimens in further malignancies.

No abstract