Pharmacologic modulation of niche accessibility via tyrosine kinase inhibition enhances marrow and thymic engraftment after hematopoietic stem cell transplantation

Abstract

Essential survival signals within hematopoietic stem cell (HSC) and thymic niches are mediated by receptor tyrosine kinases, which can be reversibly inhibited using clinically available drugs. We studied whether sunitinib, a multityrosine kinase inhibitor that inhibits KIT, enhances engraftment after bone marrow transplantation (BMT) in mice. Sunitinib diminished hematopoietic progenitor cell numbers, and sunitinib enhanced marrow, peripheral myeloid, and lymphoid engraftment after BMT in Rag1(-/-) mice. Sunitinib augmented HSC engraftment because recipients displayed increased myeloid and lymphoid engraftment and because sunitinib-treated recipients of purified HSCs showed enhanced engraftment of secondary hosts. However, sunitinib preferentially augmented T-cell engraftment with lesser effects on myeloid and HSC engraftment. Consistent with this, sunitinib preferentially depleted the early thymic progenitor subset in the thymus. Sunitinib did not increase engraftment in mice with deficient KIT signaling, and the pattern of more potent effects on T cell compared with HSC engraftment observed in sunitinib-treated hosts was also observed after BMT into KIT(W/Wv) mice. These results implicate KIT as a critical modulator of thymic niches. We conclude that transient, pharmacologic inhibition of KIT enhances accessibility of marrow and thymic niches, and provides a novel, noncytotoxic approach to accomplish engraftment after stem cell transplantation.

Figure 1

Sunitinib treatment inhibits KIT signaling in the BM and decreases BM progenitors. (A) Representative flow cytometry plots showing diminished frequencies of Lin⁻KIT^hi marrow progenitors in WT mice 4 hours after the fourth daily dose of vehicle (top panel) or sunitinib (bottom panel). (B) Frequency (top panel) and absolute numbers (bottom panel) of total marrow Lin⁻KIT^hi cells (represented by the total of all 3 stacked bar graphs) are reduced after sunitinib therapy administered as in panel A (mean percentage ± SEM: 1.55 ± 0.1 after sunitinib vs 1.03 ± 0.08 after vehicle, P < .001; and mean absolute number ± SEM: 19.2 ± 0.75 × 10⁴ after sunitinib vs 11.42 ± 0.74 × 10⁴ after vehicle, P < .001). Within the Lin⁻KIT^hi population, frequency and absolute numbers of MPPs and Lin⁻KIT^hiSCA-1⁻ progenitors are significantly reduced by sunitinib, whereas changes in HSC frequency and absolute numbers are not significant (P values shown). Data represent pooled results from 4 independent experiments; N = 18 to 20/group. (C) BM cells were harvested from untreated WT mice and then treated with sunitinib (100nM) or vehicle for 2 hours followed by recombinant mouse stem cell factor (250 ng/mL) for 30 minutes; then immunoprecipitation for KIT was performed and blotted with antiphosphotyrosine (left). Densitometric normalized ratios of phosphotyrosine/KIT are shown (right). This experiment was repeated 3 times with similar results. (D) KIT^W/Wv recipients and WT littermates received sunitinib or vehicle as described in panel A. Differences between sunitinib- and vehicle-treated KIT^W/Wv mice were insignificant (P = .18), whereas sunitinib significantly decreased the number of Lin⁻BM progenitors in wild-type littermate controls (P < .001); n = 10 mice per group. Similar results were seen in 2 different experiments. ***P < .001.

Figure 2

Sunitinib enhances marrow and peripheral myeloid engraftment after BMT into Rag1^−/− recipients. CD45.2⁺Rag1^−/− recipients received daily doses of sunitinib or vehicle on days −3 through 0, then 5 × 10⁶ TCD BM cells from WT CD45.1⁺C57Bl/6 mice on days 0 and 1. (A) Representative flow cytometry plots of gated Lin⁻ marrow on day 28 show increased donor-derived progenitors in sunitinib recipients. (B-C) Mean donor-derived marrow Lin⁻ cell frequencies and splenic myeloid cells from groups of Rag1^−/− recipients treated as in panel A and studied at serial time points. (B) Sunitinib-treated recipients showed significantly increased proportions of donor-derived Lin⁻ marrow progenitors compared with vehicle controls at all time points. (C) Significantly increased donor-derived CD3⁻B220⁻CD11b⁺ myeloid splenocytes on day 28 and 84 and significantly increased donor-derived CLPs (D), CMPs (E), CD150⁺ BM cells (F), and LSKFlt3⁻CD150⁺ BM cells (G) on day 28. *P < .05. **P < .005. ***P < .001. Radiation therapy composed Rag1^−/− recipients conditioned with 1000 cGY before BMT as controls. (B-C) Day 14, n = 4 or 5 mice/group; day 28, n = 8 to 13 mice/group; day 84, n = 4 to 15 mice/group. Data represent pooled results from 2 independent experiments. Experiments were repeated 2 to 5 times with similar results. (D-F) n = 10 mice/group; only day 28 data are available for the cell subsets.

Figure 3

Enhanced engraftment by sunitinib is transferable to secondary recipients. CD45.1⁻CD45.2⁺ Rag1^−/− recipients received sunitinib or vehicle as described in Figure 1A, followed by transfer of 3.3 × 10⁴ electronically sorted HSCs from CD45.1⁺CD45.2⁻ C57Bl/6 mice on day 0. (A) Sunitinib recipients showed an increased percentage of donor-derived Gr-1 peripheral blood cells compared with vehicle recipients on day 25; N = 4 mice/group. (B) Sixty days after BMT, whole BM from recipients described in panel A was transferred to lethally irradiated CD45.1⁻CD45.2⁺ C57Bl/6 mice. (C) Frequencies of CD45.1⁺ cells in the recipients of the secondary transplantation are shown 7 months after the secondary transplantation: n = 8 for sunitinib group; n = 5 for vehicle group. Similar results were seen on day 32 and day 63 after the secondary transplantation. *P < .05. **P < .001.

Figure 4

Sunitinib treatment decreases thymic progenitors and enhances thymic and peripheral lymphoid donor chimerism after BMT. (A-B) WT mice received sunitinib or vehicle as described in Figure 1A, and thymi were analyzed on day 4. (A) Representative flow cytometry plot showing diminished frequencies of DN1 thymocytes and thymic ETPs in sunitinib- (bottom panel) versus vehicle- (top panel) treated mice. (B) Sunitinib did not significantly decrease the percentage of CD44⁺CD25⁻ DN1 thymocytes (represented by total of stacked bar graphs), but it did induce a selective, significant decline of the KIT⁺ ETP (white stacked bar graph) within the DN1 population. Data represent pooled results from 4 independent experiments; n = 20 mice/group. (C-F) Rag1^−/− recipients received sunitinib or vehicle for 4 days, followed by transfer of 5 × 10⁶ TCD congenic BM cells from WT C57Bl/6 mice on days 0 and 1. On day 28 after BMT, sunitinib recipients showed increased numbers of donor-derived Lin⁻ thymocytes (C), increased thymic weight (D), increased total thymocyte numbers (E), increased numbers of donor-derived splenic CD3⁺ (F), and splenic B cells (CD3⁻B220⁺MHCII; G) compared with vehicle controls. Radiation therapy composed Rag1^−/− recipients conditioned with 1000 cGY before BMT as controls. Data represent pooled results from 3 independent experiments. In scatterplots, each shape represents a mouse from day 28 of panels C and F. Day 14, n = 4 or 5 mice/group; day 28, n = 8 to 13 mice/group; day 84, n = 4 to 15 mice/group. Data represent pooled results from 2 independent experiments. Experiments were repeated 2 to 5 times with similar results. *P < .05. **P < .005. ***P < .001.

Figure 5

Sunitinib enhances engraftment in immunocompetent hosts but does not enhance engraftment in KIT^W/Wv mice. (A-B) C57Bl/6 CD45.2 recipients received 4 daily doses of sunitinib or vehicle followed by transfer of 5 × 10⁶ congenic CD45.1 TCD BM cells on days 0 and 1. Organs were harvested and analyzed by FACS on day 28. (A) Representative flow cytometry plots of Lin⁻-gated thymocytes from vehicle- (left) versus sunitinib- (right) treated recipients. (B) Sunitinib recipients showed significantly increased Lin⁻ thymic and splenic T-cell engraftment compared with vehicle controls. (C) C57BL/6 × C3H.SW (H-2^b) (F1) recipients received 4 daily doses of sunitinib or vehicle followed by transfer of 5 × 10⁶ congenic CD45.1 TCD BM cells on days 0 and 1. Organs were harvested and analyzed by FACS on day 28. Sunitinib significantly increased BM multipotent progenitor (P = .03) and peripheral T-cell (P = .03) engraftment; n = 4 mice per group. (D) C57Bl/6 CD45.2 recipients received 4 daily doses of sunitinib or vehicle followed by total body irradiation with the doses indicated on day 0 and transfer of 5 × 10⁶ congenic CD45.1 TCD BM cells on days 0 and 1. On day 28, sunitinib-treated mice show increased BM engraftment compared with vehicle controls. This difference was significant at 400 cGy; n = 5 mice/group. (E) KIT^W/Wv recipients received 4 daily doses of sunitinib or vehicle followed by transfer of 5 × 10⁶ CD45.1 TCD BM cells on days 0 and 1. Sunitinib did not significantly increase the frequency of BM cells (P = .93), splenocytes (P = .70), or thymocytes (P = .69) 28 days after BMT. Data represent pooled results from 2 independent experiments; n = 13 or 14 mice/group. *P < .05. ***P < .001.