C3aR1 Contributed to Acute Myeloid Leukemia Acquired Resistance to ABT-737 and Involved In the Process of AML

Abstract 1435

Abstract

ABT-737 is a small molecule antagonist of BCL-2, BCL-XL, and BCL-w currently under evaluation in clinical trials in the oral form of ABT-263 (Navitoclax).

Acquired resistance to BCL-2 inhibitors will inevitably emerge. Published pre-clinical data showed increased levels of BFL-1 and/or MCL-1 proteins in lymphoma, and concurrent up-regulation of BCL-XL and BCL2A1 in chronic lymphocytic leukemia, which are not targeted by ABT-737.

To investigate potential mechanisms of resistance to BCL-2 inhibitors in acute myeloid leukemia, we developed resistant cell lines by long-term culture of HL-60 and MV4-11 cells with ABT-737, designated as HL-60R and MV4-11R.

Parental lines, HL-60 and MV4-11 which were highly sensitive to ABT-737 with IC50 values of 30 nM and 90nM respectively, whereas those for HL-60R and MV4-11R were 10μM and 4.7μM respectively. Annexin V binding assay revealed that both resistant lines were resistant to ABT-737 induced apoptosis as compared to the parental HL-60 and MV4-11 cells.

To explore the common pathway mediating acquired resistance to ABT-737, genes differentially expressed 2-fold or greater between parental and resistant lines on HL-60 and MV4-11 were studied. Interestingly, significant changes for BCL-2 family members were not found, suggesting that non-BCL2 family genes could play important roles in mediating the drug resistance to ABT-737. Among them, C3aR1 [Complement component 3a receptor 1] was significantly over-expressed in both resistant cell lines when compared to parental lines and verified with real-time RT-PCR.

C3aR1 inhibitor, SB 290157, showed preferential cytotoxicity to both HL-60R and MV4-11R in a dose-dependent way and spared parental cells during 48 hrs in vitro MTT assay. Our findings suggest C3aR1 plays a key role in the resistance phenotype to Bcl-2 inhibitor and blocking C3aR1 revert the resistance.

C3aR1 encodes a G-protein coupled seven trans-membrane receptor for C3a, an important inflammatory mediator. The C3a-C3aR axis modulated SDF-1–CXCR4 axis-dependent responses and regulated the homing of hematopoietic stem/progenitor cells into bone marrow. C3aR1 over expression has been associated with FLT3 and D835/I836 mutation cytogenetically normal acute myeloid leukemia, renal cancer and melanoma.

We further investigate the role of C3aR1 in 138 cases of AML including 50 cases in initial diagnosis, 68 cases in remission and 20 cases in relapse with real-time RT-PCR. The result showed that C3aR1 expression is highest in initial stage and lowest in remission (p=0.006), there was no significant difference between initial and relapse stages (p=0.384). [Table 1] For newly diagnosed AML patients, high C3aR1 is statistically associated with younger age [median: 34 vs 43 years old], higher presenting WBC [median: 39K vs 27K], higher marrow % blasts [70 vs 55%], but not related to efficacy of first induction treatment, FAB classification or conventional chromosome risk groups.

The biological significant of C3a-C3aR axis in AML and resistance phenotype is intriguing and may suggest a novel mechanism of evading the apoptotic regulation by Bcl-2 gene family as suggested by our current study. Ongoing studies will further elucidate relationship of C3a-C3aR axis in AML.