Combining chemo drug with hypoxia could offer new weapon against increasingly treatment-resistant leukaemia

Hypoxia - or low oxygen levels in bodily tissues - was thought to have a protective effect on cells affected by acute myeloid leukaemia, but a new piece of research suggests that the picture isn’t quite so straightforward.

According to one undergraduate studying at the University of the West of England, low doses of one particular chemotherapy medication can actually be more effective against leukaemia cells in hypoxic conditions. The discovery by Gabriel Hugh Gibson opens up the possibility that hypoxia could be used as part of the medical practitioner’s weaponry against leukaemia.

Gabriel’s research has just earned him an undergraduate/MSc poster prize at the first ever Minoritised Life Scientists Future Forum which attracted more than 500 delegates to the ICC in Birmingham on March 31 to April 2.

The conference brought together like-minded individuals from across the life sciences sector, higher education, and STEM, including metronics, medical robotics and AI technologies, to foster connections, champion equity, diversity, and inclusion, and showcase the incredible talent and richness of the community.

“My research investigated the effect of hypoxia on common chemotherapy treatments for acute myeloid leukaemia (AML), under the supervision of Dr Liana Gynn,” Gabriel said. 

“We found that low-dose daunorubicin (DNR) had increased cytotoxicity on leukaemic (HL-60) cells in hypoxia, which does not support the consensus that hypoxia protects leukaemic cells. 

“This outcome supports emerging evidence that low-dose daunorubicin interrupts the pro-leukaemic effects of HIF-1a, a protein induced by hypoxia that increases cell survival. This holds promising implications for future treatment developments for AML, a cancer that still largely relies on regimens established in the 1970s, despite increasing drug resistance.”

AML is the most common adult leukaemia, with an incidence that has risen by 82.2% from 1990 to 2021. AML is aggressive, with approximately 40% of patients showing resistance to the commonly used chemotherapeutics cytarabine (ara-C) and DNR. 

“AML arises in the bone marrow, which contains a microenvironment that supports homeostasis via the regulation of resident haemopoietic stem cells, their related progeny, and other interacting cells. These factors regulate the quiescence, proliferation, and differentiation of cells in the BM by controlling the expression of specific genes and proteins,” Gabriel explains.

The study investigated the effect of hypoxia, representing the endosteal niche, on the cytotoxicity and genotoxicity of ara-C and DNR in HL-60 cells. It also studied the effect that hypoxia had on the P-gp-mediated efflux mechanism by which leukaemic stem cells expel chemotherapeutics. Gabriel used a Don Whitley Hypoxystation to incubate cells at 1% O2 and compared treatments or P-gp efflux rate and competency against cells incubated at 21% O2. 

“To measure the cytotoxic effects of the chemotherapeutics, we used the trypan blue and CellTiter-Glo® assays. The trypan blue assay showed that the IC50 of each drug lowered substantially, but not significantly, in hypoxic cells. This was our first indication of a novel effect not yet described in the literature,” he says.

“Looking at the CellTiter-Glo® data, a clearer picture emerged. In low-dose (0.005 µM) DNR-treated hypoxic cells there was a significant reduction (P = 0.0001[GG1] ) in cell viability when compared to normoxic cells. The CellTiter-Glo® assay measures ATP production as a key biomarker of cell viability, which could be influenced by hypoxia-induced cell senescence. 

“However, the dose-dependent response from DNR provides enough evidence to warrant further research to confirm this possible effect. Cell cycle analysis kits and flow cytometry would verify if cell metabolism indeed played a factor by using fluorescent dyes to label specific phases of cell cycles in hypoxic HL-60 cells.”

To measure the genotoxic effects of the chemotherapeutics, Gabriel used the micronucleus assay to study cellular abnormalities in treated HL-60 cells. 

“We found that hypoxia increased the genotoxic effects of low-dose DNR, as measured by micronuclei (P = 0.045) and lobed/notched cell incidence (P = 0.041[GG1] ),” he says. 

“These results are promising, but we only incubated HL-60 in their respective oxygen conditions for 24 h after treatment. HL-60 cells require up to 48 h to complete a cell cycle, so we may not have measured the true effect of hypoxia on the chemotherapeutics, which act by interfering with cell metabolism. Future studies using the seahorse assay to measure oxygen consumption and show mitochondrial oxidative phosphorylation would confirm if 24 h is sufficient to cause a hypoxic shift in HL-60.

“We used the rhodamine-123 assay to measure how hypoxia affects the efflux of the fluorescent dye rhodamine-123 via P-glycoprotein, the same mechanism by which leukaemic stem cells efflux ara-C and DNR in multi-drug-resistant AML phenotypes. 

“We found that hypoxia did not affect the P-glycoprotein-mediated efflux mechanism of HL-60 cells. This was expected because the clonal selection of MDR1-upregulated cells occurs after initial treatment, but untreated cells remain drug-sensitive. Future investigations using drug-resistant cell lines like AML-2 or D100 may provide a model for measuring hypoxia’s effect on P-glycoprotein-mediated treatment resistance in AML.

Confirming if low-dose DNR has an increased effect in hypoxia would open the possibility for future treatment applications in AML, Gabriel said. 

DNR is a topoisomerase II poison, and he proposes that DNR-induced supercoiling of leukaemic stem cell DNA blocks the leukaemic hypoxia response element from being bound by HIF-1a, increasing treatment susceptibility in hypoxic cells, adding that this is supported by emerging evidence in pre-print that low-dose DNR inhibits HIF-1a but our finding has otherwise received no previous attention in the field.

“Currently, the most used treatment for AML is the 7+3 regimen, established by Yates et al. in 1973, and improvements in treatment have not been widely implemented. CPX-351, a formulation of liposomal 5:1 ara-C and DNR, is an approved chemotherapeutic for AML that improves patient outcome - however there is further opportunity for improvement,” he said.

“Future research to produce liposomes that contain low-dose DNR and target hypoxia via HIF-1a could prove efficacious in treatment-resistant AML patients. This outcome could take new steps towards tackling the rising treatment resistance in AML patients,” Gabriel said.