Reversible TET Inhibitor As a Hematopoietic Stem Cell Booster: A Novel Strategy to Improve Stem Cell Function
Authors: Kongkiatkamon Sunisa , Guan Yihong , Tiwari Anand , Maciejewski Phillip J , Grabowski Dale , Vatolin Sergei , Adema Vera , Kerr Cassandra M , Nagata Yasunobu , Co Milo , Snider Christina , Awada Hassan , Durrani Jibran , Parker Yvonne , Lindner Daniel J. , Sekeres Mikkael A. , Visconte Valeria , Phillips James G , Maciejewski Jaroslaw P. , Jha Babal K. 
Published: November 2019

Hematopoietic stem cells (HSCs) are responsible for the adaptation capacity in times of need but are also subjected to disease processes, natural or iatrogenic damage, and age-related attrition. The latter can lead to deficient production, decreased compensatory capacity and degenerative diseases such as MDS. In childhood, hereditary BMF predominates but increasingly with age, acquired idiopathic AA is a leading cause of HSC failure. Throughout life iatrogenic cases and cumulative exposures to environmental toxicities may lead to failure of the HSC compartment. Pharmacologic HSC boosters capable of expanding HSCs would have a wide range of clinical applications in acquired and inherited BMF states, including reconstitution of exhausted hematopoiesis after chemotherapy, aging, or HSC grafting. Currently, hematopoietic growth factors (HGF) are used but lead to progenitor rather than HSC expansion despite their success in clinical application.

Depletion of TET2 in murine models leads to impairment of cellular differentiation and increases the proportion of HSCs and progenitors suggesting that TET2 is a key regulator of hematopoietic homeostasis and HSC self-renewal. Alterations of TET2 via somatic mutations and/ or deletion are frequent in MDS whereby HSC and progenitor expansion may be a key component of neoplastic evolution. We hypothesized that chemical agents reversibly inhibiting TET2 activity might phenocopy HSC expansion due to mutations and be applied as HSC boosters.

Using structure guided approaches, we designed, synthesized and subsequently optimized bioavailable TET inhibitors (TETi). Among them, the one of the most effective TETi, named TETi76, showed dose-dependent inhibitory activity against TET dioxygenases in in vitro cell-free and cell culture systems with 5hmC production as a read out. We developed esterified forms of TETi76 and used in our cellular experimental models. Esterified TETi is bioavailable and non-toxic to normal bone marrow (BM) cells in therapeutically effective doses. Treatment of TETi76 resulted in a 44+15% and 42+17% increase in the clonogenic potential of human and murine BM cells consistent with the proliferative advantage gained by loss of TET activity in HSCs. We then performed serial replating experiments to determine the effect of TET inhibition on immature hematopoietic progenitor cells. Over 3 consecutive passages, TETi76 treatment prolonged the durability and capacity of human HSCs to maintain colony-forming cells (155+24 vs.107+14 colonies per 1x105 P1 cells). As long-term culture initiating cells (LTC-IC) are the best in vitro surrogates of HSCs, we also investigated the effect of TETi76 in LTC-IC cultures (n=3). The weekly addition of 1μM-TETi76 resulted in nearly 2-fold increase in LTC-IC numbers at the end of the culture (116±27 vs. 64 ±26 colonies per 2x106 P1 cells, p=.011). Expansion of grafts e.g., in the setting of umbilical cord HSC transplant (UCHSC) could be an important medical area of application of TETi76. We performed suspension cultures (n=3) with an optimal cocktail of hematopoietic growth factors (HGF) in the presence or absence of TETi76 (1μM). In control cultures, total cellular output peaked on day 14, but in the presence of TETi, growth continued beyond day 28. Cumulatively, total cellular output per 106 input was 27+2.61 x106cells/mL in control cultures and 32+0.642 x106 cells/mL in TETi treated cultures on day 28. CD34+ cell output was significantly higher in cultures treated with TETi vs. vehicle (2.5x105vs. 0.9X106)CD34+ cells per 1x104 CD34+ cell input). Similar effects were observed in murine BM suspension cultures. BM cells from C57BL/6 mice (n=3) were supplemented with HGF± TETi76 (1μM). TETi treatment led to a 5-fold HSC expansion compared to vehicle treated cells at 20 days of culture. The effect of TETi76 was reversed by treatment with ascorbic acid (50 μM), a known TET activator. Cumulatively our in vitro results suggest that the presence of TETi prevents exhaustion of immature cells, observed with growth factor driven expansion.

In summary, our study indicates that novel agents modulating TET activity prevent exhaustion of HSC and may help expand the HSC in vitro. In vivo experiments examining the effects of TETi on hematopoietic recovery following radiation-induced aplasia, and competitive transplant experiments of grafts exposed in vivo and in vitro to TETi are underway.

Disclosures

Sekeres: Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.