Functional Screening for Pathways Regulating Chemoresistance and Sensitivity in Leukemia Stem Cells
Authors: Mintzas Konstantinos , Kloos Arnold , Gabdoulline Razif , Schottmann Renate , Danisch Simon , Stripecke Renata , Ganser Arnold , Thol Felicitas , Heuser Michael 
Published: October 2019

INTRODUCTION

On average 5 recurrent mutations are present in each patient with acute myeloid leukemia (AML). Many mutated genes are implicated as tumor suppressor genes, but their contribution to leukemia stem cell (LSC) survival and chemoresistance is often unknown. We hypothesized that ectopic expression of the wildtype sequence of these genes will restore the function of the tumor suppressor gene and will lead to reduced clonal expansion and increased chemosensitivity.

AIM

To evaluate the contribution of recurrently mutated genes to leukemia stem cell survival and chemoresistance in human AML cells in vitro and in vivo.

METHODS

We performed a loss-of-function screening in primary human AML cells and cell lines by lentiviral expression of a pool of 22 wildtype genes associated with AML pathogenesis, which can restore gene function of a repressed pathway or a dysfunctional tumor suppressor gene. The 22 full-length cDNAs were labelled with a genetic barcode, which can be amplified with a common primer for all 22 genes. The readout of the screening was reduced representation of the barcode DNA after in vitro culture or in vivo growth in patient-derived xenograft (PDX) models, which was amplified from DNA and quantified by next-generation sequencing (NGS).

Five PDX models with favorable, normal or complex cytogenetics and 3-5 recurrent mutations per model were screened in order to identify cDNAs that could potentially limit the proliferative capacity of LSCs in vivo (5 mice per model). Nine to 16 weeks after transplantation, DNA from blood, bone marrow and spleen were analyzed by NGS. We also screened two CD34+-enriched cord blood samples in vitro. After transduction with the cDNA pool, cells were cultured for 11 days and the barcode representation was analyzed by NGS at days 2, 4, 7, 9 and 11.

Finally, we screened the human leukemia cell lines U937and PB14, a newly established cell line from an AML patient with mutations in FLT3, NPM1, RAD21, GSE1, and ROBO2. Cells were treated with cytarabine, doxorubicin or venetoclax for a 3-day period, followed by a 4-day recovery period to allow outgrowth of resistant clones and accumulation of cDNAs that conferred drug resistance.

RESULTS

Our loss-of-function screening with overexpressed wildtype genes revealed that expression of ETV6 and PTPN11 depleted LSCs in 3 of 5 PDX models and that expression of CEBPA and KDM6A depleted the progeny of normal CD34+ cells in cord blood. ASXL1, EZH2, CUX1, SMC1A and SMC3 had a general negative effect on stem cell self-renewal in both leukemic and normal CD34+ cells.

Relative frequencies of the leukemia-specific genes ETV6 and PTPN11 were reduced 3 to 16-fold and 2 to 3-fold, respectively. Both genes were not mutated in the 3 patients' diagnostic samples, but had reduced RNA expression by 10-70% compared to healthy control peripheral blood mononuclear cells. Relative frequencies of cord blood-specific CEBPA was reduced 4-fold and KDM6A 5-fold.

We then evaluated whether activation of a repressed pathway can increase sensitivity to cytarabine, doxorubicin or venetoclax in U937 and PB14 cells after 1 or 2 weeks of treatment. All three drugs showed better cytotoxic effects upon p53 expression in both cell lines by a factor of 1.4 to 2.5 fold. Cytarabine and venetoclax improved elimination of leukemic cells that had been transduced with U2AF1 in PB14 cells, which are U2AF1 wildtype. Venetoclax improved elimination of U937 cells that had been transduced with ETV6 or KDM6A, which are wildtype for these genes, while RNA expression was reduced more than 50% in these cells compared to other leukemic cell lines (NB4 and MV4-11).

CONCLUSION

Functional cDNA screening in PDX models in vivo is feasible and can reveal selective vulnerabilities of leukemic compared to normal stem cells. Our approach was validated by the finding that p53 expression improved chemosensitivity for all drugs tested in two leukemia cell lines, which is expected from the known function of p53 as a critical tumor suppressor gene.

Similarly, overexpression of the transcriptional corepressor ETV6 in leukemia cells with low ETV6 expression was found to inhibit leukemia stem cell proliferation in vivo and to sensitize U937 cells to venetoclax. Therefore, activation of ETV6 should be explored as a novel strategy to inhibit LSCs and improve treatment response.

Disclosures

Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Tetralogic: Research Funding; Sunesis: Research Funding; Daiichi Sankyo: Research Funding; Karyopharm: Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.