Clarke E¹., Traynor-Kaplan A²., Society of Toxicology 51st Annual Meeting & ToxExpo 2012, Poster Abstract No.218. ¹ReachBio, Seattle, WA, ²ISM Therapeutics, Seattle, WA.

 INTRODUCTION

Bone marrow (BM), comprising stem and progenitor cells as well as marrow stromal/mesenchymal cells (MSC), is extremely susceptible to radiation damage. BM from matched donors is often used to "rescue" patients following medical radiation for leukemia. However, in the event of accidental/environmental radiation exposure, the ability to identify appropriate donors in a timely manner is rarely possible. Using well-characterized Colony Forming Cell (CFC) assays, we evaluated a small molecule compound (ISMTR-283) on human myeloid, erythroid and mesenchymal lineages for its potential to protect the marrow progenitor cells and microenvironment from radiation damage.

MATERIALS AND METHODS

Human BM cells were ficolled to generate a mononuclear cell population and treated with 0.1 - 0.4 µM ISMTR-283 for 2 hours and then subjected to 2 or 4 Gy ionizing radiation using a RS 2000 Biologic X-Ray lrradiator (Rad Source).

To assess the protective effects of the test compound on progenitors of the myeloid (CFU-GM) and erythroid (BFU-E) lineages, cells were mixed with ColonyGel™ 1102 methylcellulose-based medium (ReachBio) and clonogenic progenitor assays were set up in triplicate in 35 mm dishes and incubated for 14 days at 37°C, 5% CO_2. Following this time period, the cultures were assessed and colonies were scored by trained personnel. Representative photographs of these colonies are presented in Figure 1.

To assess the protective effects of the test compound on human BM-derived mesenchymal cells (CFU-F), the mononuclear cells were divided into a number of identical aliquots. Two of these aliquots were pre-treated with 0.1 - 2 µM ISMTR-283 for 2 hours and then subjected to either 2 or 4 Gy ionizing radiation. Two aliquots were subjected to 2 or 4 Gy ionizing radiation and 2 hours after the radiation they were treated with 0.1 - 2 µM ISMTR-283. A final 2 aliquots were subjected to 2 or 4 Gy ionizing radiation and then 24 hours after the radiation they were treated with 0.1 - 2 µM ISMTR-283. To assess mesenchymal (CFU-F) progenitor proliferation, cells were plated in an optimized liquid based mesenchymal cell medium (ReachBio) and cultured in tissue culture treated flasks for 12 days at 37°C, 5% CO_2. Following the incubation, the medium was discarded, the flasks were washed with phosphate buffered saline, fixed with methanol and stained with Giemsa. Scoring of the colonies was then performed by trained personnel. Representative photographs of these colonies are presented in Figure 1.

Figure 1 Representative Images of Erythroid (BFU-E), Myeloid {CFU-GM) and Mesenchymal (CFU-F) Colonies

RESULTS

Ionizing radiation causes bone marrow damage and this can be seen in in vitro CFC assays in the reduction of the precursors of white blood cells (CFU-GM), red blood cells (BFU-E) and the marrow microenvironment/stroma cells (CFU-F). The effect of 0.1 - 0.4 µM ISMTR-283 on the  erythroid and myeloid progenitor proliferation is presented in Table 1 and Figure 2. At 0.4 µM, ISMTR-283 demonstrated a protective effect on BFU-E and CFU-GM proliferation following radiation exposure of 2 Gy but not 4 Gy (Figure 2).

The effect of ISMTR-283 on mesenchymal progenitors is presented in Table 2 and Figures 3 and 4. There were differences in the protective effect of the compound with time. The greatest protective effect was seen when the compound was administered in advance of the radiation, but ISMTR-283  also protected CFU-F proliferation when administered  2 or 24 hours post radiation. These data suggest that when cells are exposed to a lower level of radiation (2 Gy), ISMTR-283 protects better when it is administered in advance of the radiation, whereas when the cells are exposed to a higher level of radiation, the compound protects better if administered 24 hours after radiation exposure.

Table 1 ISMTR-283 Protects Erythroid and Myeloid Progenitor Proliferation 2 Gy but not 4 Gy Ionizing Radiation

Figure 2 ISMTR-283 Protects Erythroid and Myeloid Progenitor Proliferation Following 2 Gy but not 4 Gy Ionizing Radiation

Table 2 ISMTR-283 Protects Mesenchymal Progenitor Proliferation Prior to and Following Ionizing Radiation

Figure 3 ISMTR-283 Protects Mesenchymal Progenitor Proliferation Prior to and Following 2 Gy Ionizing Radiation

Figure 4 ISMTR-283 Protects Mesenchymal Progenitor Proliferation Prior to and Following 4 Gy Ionizing Radiation

CONCLUSIONS

ISMTR-283 is a small molecule compound that demonstrates a protective effect against ionizing radiation on human BM erythroid, myeloid and mesenchymal progenitors. In its current format, the compound showed a greater protective effect on progenitor proliferation when cells were treated in advance of ionizing radiation exposure, however, there was significant protective effect when the cells were treated with ISMTR-283 2 and 24 hours following ionizing radiation exposure. By understanding more about the mechanism of action (studies ongoing), this or other similar structured small molecule compounds may provide a prophylactic or treatment option for accidental radiation exposure.

These studies also demonstrate the utility of CFC assays in evaluating the effects of ionizing radiation and radioprotective compounds on various bone marrow progenitor lineages.

Possible mechanisms of action of ISMTR-283

1. Facilitates DNA Repair by inhibiting/slowing cell cycle progression
2. Promotes stem cell differentiation
3. Inhibits Histone Deacetylase (HDAC)