The ALDH6A1 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population derived from the HAP1 near-haploid chronic myeloid leukemia line, featuring stable disruption of the ALDH6A1 gene. This knockout model enables loss-of-function studies of mitochondrial methylmalonate-semialdehyde dehydrogenase (MMSDH). The polyclonal format provides a heterogeneous pool suitable for pooled screening and population-level analyses without selection markers, maintaining near-wild-type physiology except for ALDH6A1 inactivation. Researchers can investigate metabolic and signaling consequences of ALDH6A1 deficiency in a genomically simplified background.
The HAP1 cell line originates from KBM-7, a male-derived CML line with a near-haploid karyotype, offering unambiguous genotype-phenotype correlations in functional genomics screens. HAP1 retains oncogenic BCR-ABL signaling and leukemic metabolic dependencies, including altered amino acid and mitochondrial metabolism. The near-haploid state reduces genetic redundancy, enhancing knockout phenotype penetrance and enabling high-resolution dissection of essential pathways.
ALDH6A1 encodes MMSDH, catalyzing the oxidative decarboxylation of malonate semialdehyde and methylmalonate semialdehyde to acetyl-CoA and propionyl-CoA, using NAD+ and CoA. This reaction connects valine and pyrimidine catabolism to the TCA cycle, regulated by transcriptional coactivator PPARGC1A and transcription factor NRF1, integrating mTORC1 and BCAA availability signals. Downstream, propionyl-CoA is metabolized by PCCB and MCEE, while acetyl-CoA enters the TCA cycle. ALDH6A1 interacts with BCAT2 and BCKDHA upstream in BCAA degradation and HIBCH in valine metabolism, constituting a key network for mitochondrial substrate utilization.
In the HAP1 leukemia context, ALDH6A1 knockout disrupts BCAA and pyrimidine-derived carbon flow to mitochondrial respiration, potentially unmasking metabolic vulnerabilities exploitable in cancer therapy. Leukemic cells frequently reprogram amino acid utilization for survival, and this model enables investigation of such dependencies. Additionally, ALDH6A1 deficiency is linked to methylmalonate semialdehyde dehydrogenase deficiency, characterized by methylmalonic aciduria and neurodevelopmental delay, making these cells a tractable in vitro system for disease modeling and therapeutic screening.
Researchers can use this model in western blotting, RT-qPCR, enzymatic activity assays, and targeted metabolomics to confirm ALDH6A1 ablation and assess metabolic flux. Mitochondrial respiration and cell proliferation assays provide functional readouts. Applications include metabolic disease modeling, genetic interaction screens exploiting near-haploid genetics, and cancer metabolism studies on BCAA dependency. For further information, please contact Ascent Research.
