Synergistic pro-apoptotic effects of xanthocillin X dimethylether (XDME) with targeted BH3 mimetics Navitoclax and Venetoclax in acute myeloid leukemia

Abstract

Xanthocillin은 항바이러스작용을 가진 다양한 곰팡이에서 추출되고, 다양한 유도체들은 세포의 성장을 억제하지만 독성으로 인한 세포사멸을 일으킨다고 알려져 있습니다. Penicillium notatum에서 추출한 Xanthocillin X Dimethylether (XDME)는 독성을 갖지 않고 급성 골수성 백혈병(AML)세포의 성장을 억제하는 것을 확인할 수 있었습니다. 임상연구에 의하면 현재 AML의 생존율은 30%에 불과하며, 지난 25년 동안 치료법이 많이 바뀌지 않아 새로운 치료법의 개발이 필요한 실정입니다. 본 연구를 통해서 XDME가 백혈병세포에 대해 가지는 효과와, XDME와 임상연구단계에 있는 BH3 유사체인 Navitoclax, Venetoclax와의 combination treatment을 통해 AML에 어떤 효과가 있는지 규명하고자 했습니다. XDME는 AML세포의 성장을 현저히 저하시킴에도 불구하고 세포 사멸이 발생하지 않는 것을Trypan blue assay로 확인할 수 있었고, Zebrafish을 사용한 독성테스트에서 독성이 없는 것을 확인하였습니다. 또한 western blot을 통해 XDME가 cell cycle 조절인자인 PCNA, cyclin D1, c-myc의 발현이 감소되는 것을 증명하였습니다. 따라서 XDME는 AML에서 세포사멸을 유발시키지 않으면서 세포의 성장을 효과적으로 억제 시킨다는 사실을 밝혔습니다. XDME가 AML에 미치는 영향을 세포형태학적인 분석을 수행한 결과, XDME에 의해 vesicle형성되는 것을 확인하였고, 이로써 ER stress 및 Autophagy와 관련이 있다고 추측할 수 있었습니다. 이는 ER stress와 관련된 GRP/Bip 단백질의 감소와 autophagy를 유발하는 CHOP인자의 증가, LC3B conversion을 통해 증명하였습니다. 이에 따라 XDME는 ER stress를 유발하여 autophagy를 초래한다는 것을 확인 할 수 있었습니다. 또한, U937 cell을 비롯한 다른 AML cell line에서도 XDME는 100nM내지 1μM의 낮은 농도로 colony 형성을 억제하는 효과를 입증하였습니다. 더욱이 현재 임상연구단계에 있는 ABT199(Venetoclax), ABT263(Navitoclax)와 XDME를 55 함께 AML에 처리했을 때, 세포를 sensitize시켜 기존 농도보다 현저히 낮은 농도로 AML 세포사멸을 유도하는 사실을 규명하였습니다. 이는 Colony formation assay, Hoechst/PI staining, Caspase 3/7 assay에 의해서도 위와 상응함을 확인하였습니다. XDME는 AML세포이외에도 만성 골수성 백혈병(CML)에서 BCR-ABL의 인산화를 감소시킴으로써, ER stress를 유발해 Autophgy를 활성화시켜 세포성장을 억제하는 것을 확인 할 수 있었고, 유세포분석을 통해서 세포사멸의 효과를 지닌다는 사실을 증명할 수 있었습니다. 결론적으로 항생물질유도체인 XDME는 nano단위의 적은 농도에서 독성을 가지지 않고 cytostatic 효능이 있음을 규명함으로써, 본 화합물을 임상연구에 적용함에 있어 부작용을 최소화하고 암세포활성을 특이적으로 억제할 수 있는 유력한 물질임을 연구를 통하여 입증하였습니다.

Xanthocillin X derivatives were originally isolated from cultures of various fungi for their anti-viral activities. Selected derivatives are known to induce cell cycle arrest but with considerable toxicity. Interestingly, xanthocillin X dimethylether (XDME), extracted from the marine microorganism Penicillium commune, showed a strong anti-proliferative effect on acute myeloid leukemia (AML) cell lines without toxicity. As AML treatment did not change over the last 25 years and as novel treatment strategies are needed in order to reduce lethality of this disease, which remains high at about 70%, we further investigated the effect of XDME on AML cells alone or in combination with clinically relevant BH3 mimetics Venetoclax and Navitoclax. First, we investigated cell cycle arrest by flow cytometry and showed an accumulation of cells in G1 with a reduced S phase as early as 24h and further increasing after 48h. Interestingly, XDME did not significantly reduce viability of cells as assessed by Trypan blue staining, thus demonstrating cytostatic without cytotoxic effects. Absence of cytotoxicity was further confirmed by zebrafish toxicity assays. In order to assess the molecular mechanisms linked to cell cycle arrest, we detected decreased expression levels of S-phase marker proliferating cell nuclear antigen (PCNA), of cyclin D1, essential for G1 to S phase transition and of c-myc by immunoblot. At a cellular level, we observed morphological changes in XDME-treated cells by GIEMSA staining. U937 cells presented significant cytoplasmic vesicle formation after 24h at 5μM. Next, we investigated the effect of XDME time- and concentration-dependently on both endoplasmic reticulum (ER) stress and autophagy, known to induce such vesicle formation. Our results show that XDME (5μM) decreased the expression of glucose regulated protein (GRP)78 after 24 hours, followed by an increase after 48 and 72h, witnessing ER stress response. In addition, both phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and expression of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) were increased in a time- and dose-dependent manner. Moreover, we did not observe splicing of X-box protein (XBP)-1. In parallel to increased CHOP expression, we observed conversion of microtubule-associated protein light chain (LC) 3-I to -II by immunoblot. As the amount of LC3-II is known to be correlated to the number of autophagosomes, we concluded that autophagic flux was induced in parallel to adaptive unfolded protein response (UPR), triggered by ER stress. From a translational point of view and to generalize our findings with U937, XDME concentration-dependently inhibited 3D colony formation of various cell lines representing essential AML subtypes (HL60, THP-1, MOLT-3 and KG1) at concentrations between 100nM and 1μM. Furthermore, XDME sensitized U937 cells against two BH3 mimetics currently under clinical investigation, namely ABT199 (Venetoclax) and ABT263 (Navitoclax), inducing synergistic apoptotic cell death, quantified by hoechst/propidium iodide (PI)-fluorescence microscopy, caspase-3/7 immunoblots and activity assays as well as by poly (ADP-ribose) polymerase (PARP)-1 cleavage. Synergistic inhibition of colony formation with U937 further confirmed the pharmacological relevance of a combination of XDME with such BH3 mimetics. To further generalize our findings, we validated XDME as an inhibitor of other myeloproliferative diseases including chronic myeloid leukemia (CML). Our preliminary data show inhibition of proliferation of K562 cells already at 1μM concomitant with cell death induction as shown by flow cytometry, induction of ER stress witnessed by modulation of CHOP expression, autophagy induction shown by time- and dose-dependent LC3-I to -II conversion proceeded by a decrease of oncogenic breakpoint cluster region (Bcr)-Abelson (Abl) kinase phosphorylation at Tyr245. Altogether, our results provide a detailed insight into the potent cytostatic effect induced by the non-toxic marine natural compound XDME at nanomolar concentrations. Results further encourage validation of its anti-leukemic capacity in animal or patient-derived xenograft models followed by clinical trials alone or in combination with targeted therapeutic agents such as BH3 mimetics (AML) or Imatinib (CML).