As the most common pediatric cancer and second most common leukemia in adults (1), resistance to current treatments and disease relapse remain a significant clinical hallmark for acute lymphoblastic leukemia (ALL) (2,3) .Therefore, novel effective and less toxic therapeutics are urgently needed.
Our lab is devoted to understanding the pathophysiology of hematologic diseases such as bone marrow (BM) failure and leukemia. In attempt to search for new chemosensitizing agents that are effective and less toxic in refractory acute lymphoid leukemia (ALL) treatment, we first performed ex vivo screen of the LOPAC1280 Library, which include 1280 pharmacologically-active compounds, using the newly established AraC-resistant Molt4-Luc2 cell line. Among the 45 potential hits identified from the Synergy H1 luminescent assay, Quinacrine (QC) was found to consistently effective in both leukemic cell lines and primary samples.
We first confirmed that QC inhibits AraC-resistant Molt4-Luc2 cell growth and induces substantial apoptosis in vitro. By employing the previously established ALL-NSGS xenograft model (4), we show that QC enhances the cytotoxicity of AraC in ALL cell lines in vivo. We then asked whether combination of AraC+QC is also beneficial in primary ALL cells. By ex vivo short-term and long-term culture, we show that QC increases AraC response on primary ALL cells in vitro, but has no effect on L-LTC-ICs. Consistently, QC enhances the cytotoxicity of AraC in primary ALL cells but fails to prolong the survival of secondary recipients in a primary ALL patient cell-xenograft model.
The observation that combo AraC+QC treatment prolonged the survival of primary transplanted recipients but exhibited limited efficacy in secondary recipients, prompted us to hypothesis that a population of niche-protected quiescent LSCs or leukemia initiating cells (LICs) might have escaped the combo AraC+QC treatment. Therefore, we proposed that chasing LSCs or LICs out of the protective BM niche could improve the cytotoxic effect of the combo AraC+QC regimen in ALL therapy. By introducing the specific Cdc42 inhibitor CASIN, a HSC mobilization agent that we recently identified (5, 6), we found that CASIN enhances the eradication of ALL LSCs by AraC+QC and prolongs the survival of secondary recipients.
It is fundamental for new ALL treatments to evaluate the effect of drug combination on normal hematopoiesis. To this end, we transplanted CD34+ cells from human cord blood into sublethally irradiated NSGS mice and tested the new combo therapy in vivo. Flow cytometry and functional analysis indicate that CASIN+QC+AraC treatment does not affect long-term normal hematopoiesis. These data prove CASIN+QC+AraC treatment as a safe regimen for leukemia therapy.
Mechanistically, QC promotes autophagic cell death, increases mitochondria ROS and inhibits constitutive NF-kB activation of AraC-resistant ALL cells. Therefore, QC may act in concert with AraC to induce ALL cell death and highlight the therapeutic potential of QC in chemoresistant ALL, as well as other leukemias.
In summary, we identified Quinacrine as a novel chemosensitizing agent through an ex vivo small molecule screen, demonstrate that QC-CASIN combination is a promising strategy to eradicate leukemia stem cells and overcome chemoresistance in ALL treatment. This work is a highly collaborative teamwork. We look forward to collaborating with more researchers and clinicians to move these novel identifications from bench to bedside and improve future leukemia therapy.
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- Liu, W. et al. Rational identification of a Cdc42 inhibitor presents a new regimen for long-term hematopoietic stem cell mobilization. Leukemia, 2019. 33(3), 749-761.
- Du, W. et al. A non-myeloablative conditioning approach for long-term engraftment of human and mouse hematopoietic stem cells. Leukemia, 2018. 32(9),2041-2046.