Novel DNA-Damaging Linker Payloads Are Active in Models of Acquired Resistance to Calicheamicin and Standard of Care

Gemtuzumab ozogamicin (GO) is an FDA-approved antibody-drug conjugate that has shown clinical benefit in acute myeloid leukemia (AML) patients but has been voluntarily withdrawn from the market in the US. GO targets CD33 on AML tumor cells and delivers its cytotoxic payload, namely calicheamicin. Despite promising initial responses to GO, many patients relapsed while on therapy, and the development of clinical resistance to GO hampers its effectiveness. Initial investigations into the mechanisms responsible for clinical resistance to GO implicated the activity of multidrug resistance proteins (MDR) as a major contributor to the lack of clinical efficacy. To better understand the molecular mechanisms that may drive resistance to the payload used in GO, as well as standard of care agents in AML, we created calicheamicin-resistant and cytarabine-resistant AML cell lines. HL60 cells were continuously exposed to increasing concentrations of calicheamicin or cytarabine until resistant populations emerged. Calicheamicin-resistant HL60 (HL60-CAL-R) cells are ~40-fold resistant to calicheamicin and cytarabine-resistant HL60 (HL60-CYTAR-R) are ~2000- fold resistant to cytarabine compared to their isogenic parental counterparts as determined by in vitro cytotoxicity assays. Initial model characterization of HL60-CAL-R suggests that continuous exposure to calicheamicin induces the acquisition of a chemo-resistant phenotype highlighted by increased expression of MDR1 and MRP1/2. Furthermore, functional assays by flow cytometry revealed high levels of efflux activity in MDR1 and MRP1/2, but not BCRP, in HL60-CAL-R as compared to parental HL60. HL60-CAL-R could be re-sensitized to calicheamicin with the addition of verapamil (MDR1 inhibitor) or reversan (MDR1 and MRP1/2 inhibitor). HL60-CAL-R shows cross-resistance to daunorubicin (MDR1 substrate), but not cytarabine (non-MDR1 substrate), supporting MDR1 upregulation as one major mechanism of resistance to calicheamicin in this cell line. HL60-CAL-R cells are also cross-resistant to GO. In contrast, HL60-CYTAR-R do not have an induction of MDR1 and MRP1/2 protein levels or activity and retain sensitivity to GO in vitro and in vivo. Initial RNA-Seq profiling of HL60-CYTAR-R cells suggest they have decreased DCK expression, the loss of which was previously shown to mediate cytarabine resistance in other models. Intriguingly, HL60-CAL-R and HL60-CYTAR-R cells retained sensitivity to a proprietary next-generation DNA alkylating- and crosslinking- agent being developed at Pfizer, cyclopropylpyrrolo[e]indolone (CPI). These data demonstrate the utility of generating and characterizing drug-resistant cell lines to uncover clinically relevant mechanisms of resistance and identify next-generation compounds that can overcome them.