Soft tissue sarcomas (STS) are a heterogeneous group of malignancies predominantly affecting children and young adults. Despite improvements in multimodal therapies, 5-year survival rates are only 50% and new treatment options in STS are urgently needed. To develop a rational combination therapy for the treatment of STS we focused on ABT-199 (Venetoclax), a BCL-2 specific BH3-mimetic, in combination with the proteasome inhibitor bortezomib (BZB). Simultaneous inhibition of BCL-2 and the proteasome resulted in strongly synergistic apoptosis induction. Mechanistically, ABT-199 mainly affected the multidomain effector BAX by liberating it from BCL-2 inhibition. The combination with BZB additionally resulted in the accumulation of BOK, a BAX/BAK homologue, and of the BH3-only protein NOXA, which inhibits the anti-apoptotic protein MCL-1. Thus, the combination of ABT-199 and BZB sensitizes STS cells to apoptosis by simultaneously releasing several defined apoptotic restraints. This synergistic mechanism of action was verified by CRISPR/Cas9 knockout , showing that both BAX and NOXA are crucial for ABT-199/BZB-induced apoptosis. Noteworthy, efficient induction of apoptosis by ABT-199/BZB was not affected by the p53 status and invariably detected in cell lines and patient-derived tumor cells of several sarcoma types, including rhabdomyo-, leiomyo-, lipo-, chondro-, osteo-, or synovial sarcomas. Hence, we propose the combination of ABT-199 and BZB as a promising strategy for the treatment of STS, which should warrant further clinical investigation.

Secreted factors play an important role in intercellular communication. Therefore, they are not only indispensable for the regulation of various physiological processes but can also decisively advance the development and progression of tumours. In the context of inflammatory disease, Y-box binding protein 1 (YB-1) is actively secreted and the extracellular protein promotes cell proliferation and migration. In malignant melanoma, intracellular YB-1 expression increases during melanoma progression and represents an unfavourable prognostic marker. Here, we show active secretion of YB-1 from melanoma cells as opposed to benign cells of the skin. Intriguingly, YB-1 secretion correlates with the stage of melanoma progression and depends on a calcium- and ATP-dependent non-classical secretory pathway leading to the occurrence of YB-1 in the extracellular space as a free protein. Along with an elevated YB-1 secretion of melanoma cells in the metastatic growth phase, extracellular YB-1 exerts a stimulating effect on melanoma cell migration, invasion, and tumourigenicity. Collectively, these data suggest that secreted YB-1 plays a functional role in melanoma cell biology, stimulating metastasis, and may serve as a novel biomarker in malignant melanoma that reflects tumour aggressiveness.

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.

Soft-Tissue Sarcomas (STS) are mesenchymal malignancies with high heterogeneity that predominantly affect children and young adults. Despite routinely applied therapy strategies including radiotherapy, surgery and chemotherapy, the five-year survival rate of metastatic STS is only 50 %. Therefore, it is of high importance to focus on possible combinational therapies for the effective treatment of all kinds of STS. For such a therapy we combined the clinically approved BH3-mimetic drug ABT-199 (Venetoclax) with the proteasome inhibitor Bortezomib (Velcade). ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 whereas the proteasome inhibitor Bortezomib is effective, e.g., in multiple myeloma. Sarcoma cell lines were incubated with ABT-199 and Bortezomib alone or in combination and apoptotic cell death was flow cytometric detected by analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate an underlying mechanism for apoptosis induction, we analyzed expression of members of the Bcl-2 family involved in the apoptosis pathway by Western Blotting and performed analogue experiments in knock-out cell lines. Combined treatment with ABT-199 and Bortezomib showed synergistic cell death induction in several sarcoma cell lines including Rhabdomyosarcoma, Leiomyosarcoma and Synovial sarcoma. Loss of mitochondrial membrane potential and Annexin V staining revealed apoptosis as the underlying cell death mechanism. Interestingly, expression of Bok, a homologue of the pore-forming effector proteins Bax and Bak, was increased in response to ABT-199 and Bortezomib. Also, expression was increased for the BH3-only protein Noxa and its anti-apoptotic interaction partner Mcl-1. Knock-out (KO) of effector proteins Bax, Bak or Bok in the sarcoma cell line SW982 reduced apoptosis induction by ABT-199/Bortezomib with the most pronounced reduction in SW982/Bax-KO. Additional shRNA mediated knock-down of Noxa in Bax-KO, Bak-KO or Bok-KO SW982 cells further reduced apoptosis compared to Noxa knock-down alone. ABT-199 and Bortezomib synergistically induce apoptotic cell death in various sarcoma cell lines concomitant with enhanced expression of the Bcl-2 proteins Bok, Noxa and Mcl-1. Hence, we suggest a mechanism in which the simultaneous inhibition of anti-apoptotic Bcl-2 by ABT-199 and the stabilization of pro-apoptotic proteins Bok and/or Noxa shift the equilibrium of BCL-2-proteins towards apoptosis. Experiments in Bax, Bak, Bok, and Noxa deficient SW982 cells indicate that Bax and Noxa are crucial for the observed synergistic effect. based on these results we propose the combined treatment with ABT-199 and Bortezomib as a new and highly promising therapy option for advanced STS. Future efforts, e.g. simultaneous knock-out of relevant Bcl-2 proteins, will unravel the detailed underlying mechanism of the observed synergistic cell death induction by ABT-199 and Bortezomib. Citation Format: Sandra Weller, Alina Muenchow, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. ABT-199 and Bortezomib synergistically induce apoptosis in soft-tissue sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6227.