In Vitro Functional Rescue of a Double MPL K39N/W272R Mutant Associated with Congenital Amegakaryocytic Thrombocytopenia (CAMT) Using Crispr/Cas9

Introduction: Thrombopoietin (Tpo) and its receptor, Mpl, are the principal regulators of early and late thrombopoiesis. Mutations in MPL can drastically impair its function and be a contributing factor in chronic thrombocytosis and in congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is a rare inherited syndrome characterized by thrombocytopenia at birth, progressing to bone marrow failure and pancytopenia. The functional impact of CAMT mutations on Mpl signaling/trafficking is yet to be determined and could be relevant to multiple hematologic malignancies. Here we report unique familial cases of CAMT presenting with a previously unreported MPL mutation: T814C (W272R) in the background of the MPL K39N (Baltimore mutation), known to cause hereditary thrombocytosis (HT).

Patients: Consanguineous parents and their eldest daughter, all heterozygous for Mpl K39N/W272R, do not present any signs of disease. Their monozygotic twin daughters presented at birth with severe thrombocytopenia (platelet counts: 12,000/L and 14,000/L), low hemoglobin levels (10.4 mg/dL and 7.8 mg/dL) and very high Tpo levels (3,650 pg/mL and 3115 pg/mL). Bone marrow smears performed 19 days after birth showed severe megakaryocytopenia (only 1 megakaryocyte (MK) seen). Bone marrow colony formation assays yielded 3 and 9 MK colonies vs 84 MK colonies/10⁵ mononuclear cells for the control, leading to a diagnosis of CAMT type I. Whole blood sequencing revealed the presence of a homozygous double MPL K39N/W272R mutation. One of the twins died after bone marrow transplant. A younger male sibling, homozygous for MPL K39N/W272R mutation, has also been diagnosed with CAMT type I.

Objectives: This study focuses on the functional characterization of the novel MPL W272R and K39N/W272R mutations and in vitro genetic engineering as a potential therapeutic option for CAMT.

Methods: Human megakaryoblastic UT-7 and murine Ba/F3 cells stably expressing human wild-type (WT) Mpl or K39N, W272R or doubly mutated K39N/W272R Mpl fused to mNeonGreen were used as models. Confocal microscopy, proliferation and surface biotinylation assays, as well as co-immunoprecipitation (co-IP) and western blotting analysis, were used to elucidate the function and trafficking of Mpl mutants. CRISPR/Cas9 genetic engineering was used to repair mutant MPL and rescue its function.

Results: Confocal microscopy shows that a significant fraction of chimeric WT Mpl protein reaches the cell surface. Significant surface expression is also noted for Mpl K39N. In contrast, the chimeric Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, showed no detectable surface expression of the Tpo receptor. These results were confirmed by surface biotinylation assay. Co-expression of WT CALR fused to RFP, used as an ER marker, showed significantly higher co-localization (Pearson's R value) with mutant Mpl than with WT Mpl, evidence that the large majority of receptors were retained within the ER. We also evaluated Tpo signaling through the JAK/STAT, MAPK and PI3K pathways and Tpo-induced proliferation. Both WT and K39N-mutated Mpl were competent for signaling, while single or double mutants bearing W272R were unresponsive to Tpo. Tpo-induced signaling was partially restored via GRASP55 over-expression (forcing ER-trapped Mpl to traffic to the cell surface). Genetic engineering performed on cells carrying the W272R mutation restored the WT sequence and the response to Tpo, with similar cell proliferation as WT Mpl cells. In addition, co-IP studies indicate that Jak2 associates strongly with WT Mpl and Mpl K39N but not with Mpl W272R.

Conclusion: We report a new mutation of Mpl (W272R) present in cis with HT-causing K39N mutation in the context of CAMT. The absence of symptoms in the Mpl K39N/W272R-mutated parents (found to be heterozygous) can be explained by the opposite (apparently neutralizing) effects of the two mutations on the trafficking and signaling of Mpl, as shown by confocal microscopy, western blotting and proliferation assays. In children homozygous for Mpl K39N/W272R, the W272R mutation prevented Mpl binding of Jak2 and expression at cell surface, rendering Tpo signaling impossible despite the presence of the K39N mutation. Function of the deficient Mpl receptor could be rescued using two separate approaches: CRISPR/Cas9 genetic engineering and GRASP55 over-expression. Cell-permeable Tpo analogs will also be tested.