Runx2 takes center stage in regulating HSC expansion Free

In this issue of Blood, Meaker et al¹ report that the transcription factor Runx2 is an important negative regulator of hematopoietic stem cell (HSC) expansion and T-cell lineage commitment ex vivo and in vivo through the use of a genome-wide CRISPR knockout (KO) screen in HSCs, improving our understanding of intrinsic factors that control HSC proliferation and cell fate.

The ability to robustly expand multipotent HSCs has long been of great interest in the field of hematopoiesis. HSC transplants (HSCTs) for a variety of blood disorders and diseases rely on a significant number of HSCs or CD34⁺ cells, and the number of cells transplanted alters the success of the procedure. For example, one study showed higher doses of CD34⁺ cells in HSCTs in patients with hematological malignancies were correlated with increased overall survival and decreased risk of transplant-related death.² Although recent advancements in ex vivo culturing systems have expanded capabilities in studying HSCs,^3,4 our understanding of the interplay between proliferation/differentiation and preservation of self-renewal is still incomplete. Meaker et al sought to identify novel regulators of HSC expansion through a genome-wide CRISPR KO screen in purified murine hematopoietic stem/progenitor cells.¹ Their screen identified Runx2 as a potent negative regulator of HSC expansion. Loss of Runx2 in ex vivo culture HSCs led to a significant increase in HSC numbers.

Three members of the Runx family of transcription factors exist: Runx1, Runx2, and Runx3. Runx1 is a critical transcription factor controlling hematopoietic cell fate, and recurrent RUNX1 mutations are known drivers in human myeloid malignancies.⁵ However, the role of Runx2 in hematopoiesis remains more of a mystery. Prior studies dissecting the function of Runx family members suggested Runx2 may cooperate with oncogenic fusions to help drive leukemia transformation through its control of progenitor differentiation potential.⁶ Therefore, the observation that Runx2 came out as a top hit in the screen performed by Meaker et al was intriguing and suggested that Runx2 may also regulate the ability of HSCs to proliferate. In vivo KO of Runx2 recapitulated their ex vivo findings, with transplanted Runx2 KO HSCs showing significantly increased chimerism in recipient peripheral blood and bone marrow compared to wild-type HSCs, which was robustly sustained through secondary transplant.

Runx2 KO increased HSC numbers with normal blood parameters in recipient mice. However, the authors identified decreased T-cell output from Runx2 KO HSCs, which mapped to the early DN1 stage of T-cell progenitor commitment. Elegant gene dosage studies found that heterozygous loss of Runx2 created haploinsufficiency, which could sustain increased HSC expansion while rescuing the T-cell differentiation defect. Furthermore, ex vivo clonal studies of HSCs discovered that Runx2 KO leads to increased self-renewing divisions of HSCs at the expense of asymmetrical divisions, which lead to downstream progenitors. Prior work in hematopoiesis has underscored the requirement for a tightly regulated balance between quiescence, cell cycle, and proliferation in determining cell fate. For instance, quiescent, noncycling subsets of early hematopoietic progenitor cells were found to harbor different lineage biases upon differentiation compared to their faster cycling counterparts.⁷ Moreover, cell cycle lengthening has been shown to increase myeloid differentiation through the buildup of specific transcription factors.⁸ Perhaps, the authors, in identifying Runx2 as a novel regulator of HSC expansion, have also uncovered a potential factor involved in the control of this balance. The results described by Meaker et al support the concept that both ex vivo and in vivo HSC expansion can be altered by perturbing transcription factor expression and/or activity. However, moving this concept to clinical application will require a lot more work. Nevertheless, the potential translational implications are exciting. In sum, further studies are needed to determine whether transient inhibition of Runx2 (and/or other factors found in the authors’ screen) can uncover novel mechanisms for HSC expansion, not only for those interested in dissecting the biology of the hematopoietic system but also for new strategies related to HSC mobilization in the clinical space.

Conflict-of-interest disclosure: Unrelated to this manuscript discussed, L.A.M. has previously received honoraria for speaking arrangements and had previously served on a speakers bureau for Mission Bio, Inc.