The Chromosome 21 Kinase DYRK1A Controls Cell Cycle Exit and Survival During Lymphoid Development and Is a Novel Therapeutic Target In Acute Lymphoblastic Leukemia

The dual specificity tyrosine-regulated kinase 1A (DYRK1A) phosphorylates a growing number of proteins to influence various aspects of cell biology, including survival, apoptosis, proliferation, and neuronal development and function. Encoded on human chromosome 21, the DYRK1A locus resides in the Down syndrome critical region (DSCR) and has been implicated in the pathogenesis of neurocognitive deficits that result from trisomy 21. We recently demonstrated a prominent tumor-promoting role for DYRK1A in acute megakaryoblastic leukemia in children with DS (DS-AMKL). This discovery prompted us to ask whether DYRK1A is important for normal hematopoiesis, and if additional types of leukemia result from enhanced DYRK1A activity.

Apart from our study in DS-AMKL, the involvement of DYRK1A in normal and malignant hematopoiesis has not been described. Dyrk1a germline knockout mice die mid-gestation; therefore, we generated conditional knockout (CKO) mice to allow inducible deletion of the kinase in hematopoietic cells with Mx1-Cre. In the bone marrow, CKO mice exhibit significant reductions of the hematopoietic stem cell-enriched LSK and common lymphoid progenitor populations (5-fold and >20-fold reductions vs. controls, respectively). In B cell lineage, CKO mice have 2-fold reductions of small pre-B and immature B cells, and a 5-fold reduction of mature recirculating B cells. In the thymus, CKO mice show dramatic loss of CD4⁺/CD8⁺double positive cells (DP, >30-fold fewer at 4 weeks). Furthermore, DYRK1A-deficient cells fail to reconstitute lymphoid cells in competitive bone marrow transplantation experiments and give rise to virtually no pre-B cell colonies in ex vivo assays. In contrast to these lymphoid defects, myeloid development is largely normal, with slightly increased numbers of erythroid precursors, granulocytes, and monocytes.

To understand the mechanisms by which DYRK1A controls lymphopoiesis, we performed microwestern blot arrays. This approach allowed us to simultaneously screen >200 antibodies to identify differentially expressed proteins between CKO and control lymphoid cells. We found increased levels of cell cycle regulatory proteins, such as CDK1, suggesting that DYRK1A modulates cell cycle status during lymphoid development. Indeed, cell cycle analysis revealed that there were 20-30% fewer quiescent (G0) cells among CKO small pre-B cells and DP thymocytes, two developmental stages characterized by exit from the cell cycle following a proliferative burst. Among its targets, DYRK1A is known to contribute to DREAM (DP, Rb-like, E2F, and MuvB) complex assembly via phosphorylation of LIN52. The DREAM complex, not previously described in hematopoiesis, promotes quiescence by repressing the transcription of numerous cell cycle-associated genes during G0. RNA sequencing analysis in flow-purified G0 cells confirmed that DREAM complex targets are markedly de-repressed in the affected CKO populations, highlighting the importance of DYRK1A/DREAM complex interaction during lymphoid cell cycle exit.

Both B- and T-lineage ALL express high levels of DYRK1A relative to other tumor types. Having observed that DYRK1A is essential for lymphoid cells, we next asked whether targeting DYRK1A with small molecule inhibitors would be an effective therapy for ALL. We found that EHT 1610, a highly selective DYRK1 inhibitor (Leblond B., Casagrande A.-S., Désiré L., Foucourt A., Besson T., DYRK1 inhibitors and uses thereof, WO 2013026806, 2013.02.28), dose-dependently induced apoptosis in B- and T-ALL cell lines and primary human pediatric ALL samples. Thus far, all cell lines and all primary samples tested were exquisitely sensitive to EHT 1610. Moreover, EHT 1610 induced apoptosis of primary ALL cells that were resistant to cytarabine, suggesting that DYRK1A inhibitors may be used in combination with standard ALL therapies for refractory or relapsed cases. Together, our data establish novel essential roles for DYRK1A in both normal and malignant lymphoid development and provide rationale for the design of DYRK1A-targeted ALL therapies.