Abstract
Casein kinase 1α (Ck1α) is a serine/threonine kinase located in the common deleted region (5q32) in del(5q) myelodysplastic syndrome (MDS). Ck1α is a regulator of the canonical WNT signaling pathway and may play a role in the clonal advantage of del(5q) cells. In addition, we identified CK1α as a therapeutic target in myeloid malignancies in an in vivo RNA interference screen, and haploinsufficiency for CK1α could further sensitize del(5q) cells to CK1α inhibition. To explore the biology and therapeutic potential of CK1α in MDS, we generated a conditional Ck1α knockout mouse model.
Conditional homozygous inactivation of Ck1α resulted in bone marrow failure, ablation of hematopoietic stem and progenitor cells, a severe anemia and rapid lethality within 7-12 days, confirming that Ck1α is essential for hematopoietic stem and progenitor cell survival. In contrast, mice with haploinsufficiency of Ck1α developed a hypercellular bone marrow, as is typical in MDS, a significantly elevated white blood cell count (p=0.002) and normal hemoglobin levels. The hematopoietic stem cells (LSK, LT-HSC, ST-HSC) as well as progenitor cells (LK, pre-GMP, GMP, pre-CFU-e, CFU-e, pre-megakaryocytes-erythrocytes) were not affected by Ck1α haploinsufficiency 14 days after induction. Only the megakaryocytic progenitor cells (p=0.04) were significantly reduced. This finding was in line with severe dysplasia and hypolobulated micromegakaryocytes observed in the bone marrow, another typical histomorphological feature of del(5q) MDS.
In long-term experiments up to 8 months, the survival of mice with Ck1α haploinsufficiency was not impaired, although we observed an exhaustion of the stem cell pool with significant reduction of ST-HSC (p<0.001), LT-HSC (p=0.003), and MPP (p=0.007). We were able to demonstrate that this significant reduction is a cell-extrinsic effect. In transplantation and HSC repopulation assays, an intact HSC function and even a significant expansion of hematopoietic stem cells and progenitor cells with Ck1α haploinsufficiency was confirmed in comparison to MxCre controls (LSK p=0.019; LK p=0.035; CMP p=0.036; GMP p=0.027; MEP p=0.005), suggesting a repopulation advantage of HSC with Ck1α haploinsufficiency. In contrast, Ck1α homozygous deletion leads to a cell-autonomous, p53-mediated HSC failure in transplantation assays.
To dissect the mechanism of hematopoietic stem cell expansion in Ck1α haploinsufficiency on the one hand and the hematopoietic stem cell ablation after Ck1α ablation on the other hand, we analyzed regulatory mechanisms including proliferation and apoptosis in LK cells (myeloid progenitor cells) and LSK cells (enriched for hematopoietic stem cells). Ablation of Ck1α led to a significant increase (p=0.001) in the number of LSK and LK in the S/M/G2 phase, accompanied by a significant reduction in the G0/G1 fraction, suggesting their exit from quiescence. Ck1α haploinsufficiency led to a significant increase in the fraction of cycling cells in myeloid progenitor cells (LK, p=0.052), the quiescent hematopoietic stem cells were not significantly affected. In Western Blots of ckit+ hematopoietic stem and progenitor cells, a significant increase of intracellular ß-catenin levels was detected in both Ck1α haploinsufficient and even stronger in Ck1α ablated cells, accompanied by an exit from stem cell quiescence shown by loss of p21-mediated growth arrest and up-regulation of phosphorylated retinoblastoma protein indicating cell cycle progression from G0 to G1 in comparison to the MxCre+ control cells. Ck1α ablation led to p53-mediated apoptosis in stem and progenitor cells (Annexin V/7-AAD). In Ck1α haploinsufficient cells, apoptosis was not significantly induced in neither LK cells or in LSK cells although p53 induction was observed in the bone marrow.
Taken together, our results indicate that Ck1α is essential for hematopoietic stem and progenitor cell survival, but that Ck1α haploinsufficiency does not decrease, and may increase, hematopoietic stem cell function. This finding highlights the potential of preferential elimination of the del(5q) hematopoietic stem cells through Ck1α inhibtion and thus provides a potential therapeutic window. Consistent with this hypothesis, targeting the haploinsufficient kinase activity in vitro with the Ck1α small molecule inhibitor D4476, selectively targets CK1α haploinsufficient cells relative to wild-type cells.
Disclosures:
Järås: Cantargia: Equity Ownership.
Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells
