Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski’s rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.

Treatment of acute myeloid leukemia (AML) remains inefficient due to drug resistance and relapse, particularly in patients with FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD). Marine-derived natural products have recently been used for drug development against AML. We show in this study that petromurin C, which was isolated from the culture extract of the marine-derived fungus Aspergillus candidus KUFA0062, isolated from the marine sponge Epipolasis sp., induces early autophagy followed by apoptotic cell death via activation of the intrinsic cell death pathway concomitant with mitochondrial stress and downregulation of Mcl-1 in FLT3-ITD mutated MV4-11 cells. Moreover, petromurin C synergized with the clinically-used FLT3 inhibitor gilteritinib at sub-toxic concentrations. Altogether, our results provide preliminary indications that petromurin C provides anti-leukemic effects alone or in combination with gilteritinib.