Coinhibition of BCL-2 and NHE1 pathways may have a potential to overcome venetoclax resistance

BCL-2, BCL-XI, along with MCL-1 proteins are known to play a significant role in tumor cell survival with anti-apoptotic properties. Venetoclax, a BCL-2 selective inhibitor, in combination with hypomethylating agents, has become the standard of care for the treatment of unfit elderly patients, and BCL-2 is considered a universal target in acute myeloid leukemia (AML). However, resistance to venetoclax may be a concern in providing durable responses. Deamidation of the intracellular pH level is also known to modulate the BCL-2 pathway. Shin Young Hyun et al. conducted a study to investigate whether inhibiting both BCL-2 pathway with venetoclax and pH through the Na/H exchanger 1 (NHE1) pathway with 5-(N, N-hexamethylene)-amiloride (HMA) concurrently would eliminate resistance to venetoclax in AML cell lines. They presented the results during the Virtual 46th Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT), and here we are pleased to summarize key points.¹

Methods

• Cell lines sensitive (MOLM13, MV4, and RS4-11), and resistant (THP1 and U937) to venetoclax were treated with different venetoclax concentrations (see Table 1)

Table 1. Venetoclax concentrations used in AML cell lines

AML cell lines		Venetoclax concentrations
Sensitive	MOLM13	1–100 nM/L
MV4
RS4-11
Resistant	THP1	10–1,000 nM/L
U937	0.1–10 µM/L

• HMA concentration ranged between 1–10 µM/L
• Annexin-V assay was used to analyze apoptotic cells, and western-blot analysis was used to evaluate the expression of BCL-2 family proteins
• Cell lines treated with venetoclax alone or the combination of venetoclax and HMA were compared

Results

In sensitive cell lines, venetoclax alone led to a concentration-dependent apoptosis. The combination was synergistically associated with higher rate of cell apoptosis, and almost complete apoptosis with lower concentrations of venetoclax and HMA, suggesting improved sensitivity to venetoclax. The difference was significant (p < 0.05) for all venetoclax concentrations in all sensitive cell lines.

In resistant cell lines, the apoptosis rate was low with venetoclax monotherapy, and higher concentrations could not induce an adequate level of apoptosis. However, combination treatment with HMA led to apoptosis in the majority of THP1 and U937 cells, 80% and 90%, respectively. For the THP1 cell line, the difference was significant (p < 0.05) for 1 nM, 10 nM, and 100 nM venetoclax concentrations, while the difference was significant (p < 0.01) with the 10 uM concentration only in the U937 cell line.

BCL-2 family protein analysis demonstrated that AML cell lines with high BCL-2 expression were venetoclax sensitive (all except U937). Overexpression of MCL-1 proteins was associated with resistance to venetoclax (THP1 and U937 cells), and when accompanied by increased BCL-2 expression, the sensitivity to venetoclax was decreased.

Conclusion

These results indicate that concurrent targeting of BCL-2 and NHE1 pathways induced sensitivity to venetoclax in all cell lines, suggesting this approach may overcome the challenge of venetoclax resistance in the treatment of AML. Further investigations are needed to elucidate the exact mechanism of this approach and its impact on signaling molecules involved in BCL-2-related pathways.