PTEN loss reprograms lipid metabolism to modulate venetoclax resistance in acute myeloid leukemia Free

Acute myeloid leukemia (AML) is an aggressive and biologically heterogeneous hematologic malignancy. It is characterized by the clonal expansion of aberrant myeloid progenitor cells and predominantly affects older adults. As many elderly patients are not suitable for intensive chemotherapy or allogeneic hematopoietic stem cell transplantation, the five-year overall survival rate remains below 10%.

The emergence of venetoclax, a novel, orally bioavailable selective BCL-2 inhibitor, provides a promising therapeutic option for elderly AML patient population. However, resistance to venetoclax develops in approximately 30% of patients, posing a major clinical challenge. Although accumulating evidence implies metabolic reprogramming in venetoclax resistance, effective strategies to reverse this process remain elusive.

To recapitulate the stepwise acquisition of venetoclax resistance, we first generated MOLM-13 sublines exhibiting intermediate and complete resistance. Whole-exome sequencing of the derived lines identified two BCL-2 mutations. Interestingly, the progressively increasing variant allele frequencies correlate with the resistance severity, implicate these mutations as potential molecular drivers of venetoclax resistance. Remarkably, a relapsed AML patient after allogeneic HSC transplantation, went under venetoclax treatment for the relapse, became venetoclax-resistant while developed the same two BCL-2 mutations in patient serial leukemic cell samples, underscoring the clinical relevance of these findings.

To elucidate the molecular alterations associated with venetoclax resistance, we firstly performed transcriptomic and proteomic profiling on the resistant MOLM-13 sublines. These analyses revealed a marked upregulation of lipid metabolism pathways in resistant cells compared to wild-type. This finding prompted us to further investigate whether metabolic reprogramming functionally contributes to resistance. To this end, we conducted a metabolism-focused CRISPR-Cas9 screen in wild-type MOLM-13 cells under venetoclax pressure. The screen identified 175 and 191 negatively selected genes at day 8 and 16, respectively, with 30 genes overlapping at both time points. Pathway enrichment analysis highlighted lipid metabolism as a key pathway, with PTEN prominently enriched, suggesting a potential role in mediating venetoclax resistance via metabolic regulation.

We next sought to validate the functional role of PTEN in modulating venetoclax response. In vitro, genetic ablation of PTEN using CRISPR/Cas9 or shRNA significantly increased venetoclax-induced apoptosis in both parental and resistant AML cell lines. Pharmacological inhibition of PTEN with small-molecule inhibitors (SF1670 or bpV(HOpic)) produced similar sensitizing effects. Importantly, these findings were corroborated in primary AML samples, where PTEN suppression enhanced venetoclax responsiveness. In vivo, PTEN inhibition in xenograft models led to increased apoptotic response upon venetoclax treatment, further confirming its critical regulatory role in venetoclax response.

Mechanistically, PTEN loss led to pronounced lipid droplet accumulation, as confirmed by MS/MS-based lipidomic profiling showing elevated fatty acid levels. ¹³C-labeled palmitate tracing showed reduced incorporation of fatty acids into the TCA cycle, indicating impaired β-oxidation. Concomitantly, upregulation of FASN and SREBP suggested increased lipid synthesis. This dual effect of reduced lipid degradation and increased synthesis resulted in intracellular lipid accumulation, which caused mitochondrial damage, as evidenced by elevated ROS levels. This mitochondrial stress may contribute to reduced resistance to venetoclax by promoting apoptosis, providing a mechanistic link between PTEN loss, lipid metabolism reprogramming, and enhanced drug sensitivity.

In conclusion, we identify PTEN as a key lipid metabolic regulator of venetoclax sensitivity in AML. By linking lipid metabolic reprogramming to apoptotic vulnerability, we uncover a mechanistic basis for venetoclax sensitization. Our findings highlight the potential of targeting lipid metabolic pathways as a strategy to overcome venetoclax resistance in AML, particularly in the context of BCL-2 mutations.

MT.C, LZ.C and YJ.J contributed equally to this work.

Corresponding authors: WP.Y, EL.J and YJ.C.