PF426 GENOTYPE‐RESPONSE CORRELATION IN DRIVE PK, A PHASE 2 STUDY OF MITAPIVAT (AG‐348) IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY (2019)

Background: Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by mutations in the PKLR gene, leading to a deficiency of the glycolytic enzyme red cell PK (PK‐R). Mitapivat (AG‐348) is an oral, small‐molecule, allosteric activator of PK‐R that activates the wild type and a range of mutant PK‐R enzymes associated with PK deficiency. In a phase 2 study in 52 patients (pts) with PK deficiency (DRIVE PK, NCT02476916), 50% of pts experienced a maximum hemoglobin (Hb) increase of >1.0 g/dL (mean maximum increase, 3.4 g/dL; range, 1.1–5.8 g/dL). Hb increases were observed in patients with a variety of PKLR mutations and were associated with the presence of ≥1 missense mutation. As PK deficiency is genetically heterogeneous (>200 described mutations), we sought to better understand the molecular parameters associated with Hb increases in mitapivat‐treated pts. Aims: To analyze the relationship between Hb increase and pt genotype, biochemical response to mitapivat, and baseline PK‐R protein level. Methods: Whole blood samples were collected from pts enrolled in DRIVE PK. Genotypes were determined by Centogene AG. Baseline PK‐R protein was quantitated by antibody‐based capture and detection using a Meso Scale assay and the signal normalized to a control sample from a subject without PK deficiency. Results: Of 106 PKLR mutations in the 52 pts (2 pts carried 3 mutations), there were 47 unique mutations, including 28 missense and 19 non‐missense. Of these, only 10 were present in ≥2 pts. Of these 10 mutations, some were more likely to be associated with Hb increases (e.g. increases were observed in 5/5 pts with ≥1 R486W mutation and 4/4 pts with ≥1 T384 M mutation). Other mutations, including V134D (0/3), E241stop (1/6), and R510Q (5/14), were more likely to be associated with a lack of Hb increase. Notably, 1/10 pts who had 2 non‐missense mutations and 0/5 pts homozygous for the R479H “Amish” mutation had Hb increases. We have previously reported that, in biochemical assays, mitapivat could efficiently activate recombinantly expressed R479H and R510Q mutant enzymes. Although both of these are technically missense mutations, it is well understood that R510Q results in severely decreased protein stability, and the R479H mutation, occurring at the splice junction between 2 exons, can deleteriously affect mRNA splicing. Therefore, as mitapivat works by directly binding and stimulating the activity of residual mutant PK‐R enzyme, we explored the hypothesis that PK‐R protein levels in pts with these mutations might be too low to allow for adequate target engagement at clinically tested doses. Pts with Hb increases had, on average, higher baseline PK‐R protein levels (49% of control vs 13% in pts without Hb increases). All 10 pts with non‐missense/non‐missense mutations had nearly undetectable PK‐R protein levels (average of 3.7% of control), consistent with and confirming the predicted effect of these mutations on protein expression. Baseline PK‐R protein levels were also lower in pts with ≥1 R510Q (18% of control) or R479H (19% of control) mutation than in pts with other missense mutations (59% of control). Summary/Conclusion: These analyses demonstrate that Hb increases upon mitapivat treatment are associated with the presence of ≥1 missense PK‐R mutant enzyme with residual protein for mitapivat to bind and activate. These data strongly suggest that the hematologic effects of mitapivat come from the proposed mechanism of action of restoration of glycolytic pathway activity in PK‐deficient red cells. image