Myeloablative hematopoietic stem cell transplantation improves survival but is not curative in a pre-clinical model of myelodysplastic syndrome

<div><p>Allogeneic hematopoietic stem cell transplantation (A-HSCT) remains the only curative option for patients with myelodysplastic syndrome (MDS). We used the NUP98-HOXD13 (NHD13) murine model for MDS to study HSCT in a pre-clinical setting. NHD13 recipients transplanted with syngeneic bone marrow (S-HSCT) following myeloablative irradiation showed disease remission, with normalization of peripheral blood parameters and marked decrease in circulating leukocytes derived from the MDS clone. Despite the disease remission and improved survival compared to non-transplanted NHD13 controls, all mice eventually relapsed, indicating persistence of a long-lived radio-resistant MDS clone. In an effort to induce a graft versus leukemia (GVL) effect, A-HSCT with donor bone marrow that was mismatched at minor histocompatibility loci was compared to S-HSCT. Although recipients in the A-HSCT showed a lower early relapse rate than in S-HSCT, all mice in both groups eventually relapsed and died by 54 weeks post-transplant. To obtain a more significant GVL effect, donor splenocytes containing reactive T-cells were transplanted with allogeneic bone marrow. Although the relapse rate was only 20% at post-transplantation week 38, suggesting a GVL effect, this was accompanied by a severe graft versus host disease (GVHD) Taken together, these findings indicate that a myeloablative dose of ionizing radiation is insufficient to eradicate the MDS initiating cell, and that transplantation of donor splenocytes leads to decreased relapse rates, at the cost of severe GVHD. We suggest that NHD13 mice represent a feasible pre-clinical model for the study of HSCT for MDS.</p></div

Effect of GVHD using the NHD13 A-HSCT model.

<p>(A) GVHD was induced by transplantation of splenocytes with BMNC. GVHD score vs time for individual recipients; the values for #227 and #235 are identical and thus the curves are superimposed on one another(B) Cumulative incidence of severe GVHD or relapse. Mice were euthanized when they displayed clinical signs of disease such as lethargy, tachypnea, or hunched posture. (C) Survival curve comparison of Non-HSCT NHD13, A-HSCT and A-HSCT + Splenocytes. Non-HSCT NHD13 refers to NHD13 transgenic mice not subjected to irradiation and HSCT.</p

Clinical outcome of A-HSCT with induced GVHD/GVL.

<p>Clinical outcome of A-HSCT with induced GVHD/GVL.</p

Overall survival and cumulative incidence of relapse.

<p>(A) Log RANK test for Non-HSCT vs S-HSCT, p = 0.0013; Non-HSCT vs A- HSCT, p = 0.0054; Allo HSCT vs Syn HSCT, p = 0.7373. Non-HSCT NHD13 refers to NHD13 transgenic mice not subjected to myeloablative irradiation and HSCT. WT-HSCT refers to WT recipient mice transplanted with WT syngeneic BMNC following myeloablative irradiation. (B) Cumulative incidence of relapse for S-HSCT and A-HSCT.</p

Syngeneic hematopoietic stem cell transplant (S-HSCT) as therapy for MDS.

<p>(A) Schematic illustration of S-HSCT experiment, using myeloablative (10 Gy) or non-myeloablative (6.5 Gy) total body irradiation as a conditioning regimen (B) Representative FACS profiles of individual recipients at 16 and 26 weeks post-transplant. CD45.2+ indicates cells derived from the host. (C) CD45.2+ cells from individual recipients; increasing CD45.2+ cells indicate relapse of host (MDS-derived) hematopoiesis. (D) Hemoglobin changes following S-HSCT. (E) MCV, Mean corpuscular volume. (F) PLT, platelet count. (G) ANC, absolute neutrophil count. (H) Survival curve after HSCT. The sword symbol indicates death of the recipient.</p

Clinical outcome of therapeutic HSCT in NHD13 mice with MDS.

<p>Clinical outcome of therapeutic HSCT in NHD13 mice with MDS.</p

Myeloid dendritic cells (CD11b⁺CD11c⁺) are increased in TDLN and are required for tumor regression.

<p>(A) C57BL/6 mice were challenged with 1×10⁶ MB49 on day 0 and treated with Val-boroPro (closed bars) or saline (open bars) during week one (n = 4/group). Tumor-draining lymph nodes were harvested and analyzed by flow cytometry on day 7 (p<0.05, Mann-Whitney) (B) C57BL/6 mice were inoculated with 10⁶ MB49 and injected with clodronate IP (0.1 mL/10 g body weight) every other day from day –1 to 9 following tumor inoculation. Control groups were injected with empty lysosomes or saline (sham). Val-boroPro was administered orally for 1 week (days 3–7). Survival was significantly different in Val-boroPro treated mice given clodronate (thick solid line) compared to those treated with Val-boro-Pro and given empty lysosomes (gray solid line) (*p<0.05, Logrank test, n = 5/group). (C) CD11c-diphtheria toxin (DT) chimeric mice were generated by transplanting bone marrow from CD11c-DT transgenic mice into lethally irradiated C57BL/6 recipients (n = 5/group). Female chimeras were inoculated with 10⁶ MB49 on day 0 and treated with Val-boroPro or saline during week one (days 3–7) with or without IP injections of DT (8 ng/1 g body weight) every other day from day –1 to 9. Tumor volumes were significantly larger in Val-boroPro treated chimeric mice receiving DT (closed circles) compared to Val-boro-Pro treated chimeric mice receiving saline (closed squares) (p<0.001 for all timepoints beyond day 10). Tumor volumes were not statistically different between saline treated (open squares) and DT treated (open circles) chimerics not treated with Val-boroPro. Experiments displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g007" target="_blank">Figures 7A–C</a> were each conducted twice.</p

Val-boroPro treatment is associated with an acceleration of tumor-induced priming.

<p>(A) Female mice were inoculated with MB49 and treated with Val-boroPro (closed bars) or saline (open bars) (n = 12/group). Spleens and lymph nodes were harvested from mice on days 10, 17, and 24 for IFN-gamma ELISPOT analysis (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney). (B) Mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g003" target="_blank">Figure 3A</a>. On day 17, greater numbers of HY-reactive CD8⁺ T cells were observed in the lymph nodes of saline-treated (open bars) mice compared to Val-boroPro treated (closed bars) mice (*p<0.05, **p<0.01, Mann-Whitney, n = 5/group). (C) Composite data showing combined (UTY+SMCY+DBY) relative to tumor volume from mice treated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">figure 1A</a> with saline (open squares top, open bars bottom) or Val-boroPro (closed squares top, closed bars bottom) (n = 12/group). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g003" target="_blank">Figures 3A–C</a> are representative of 3 experiments.</p

Val-boroPro mediated tumor regression is T cell dependent.

<p>(A) Female C57BL/6 mice were challenged on day 0 with 1×10⁶ MB49 and treated with 20 µg Val-boroPro during weeks 1 through 4 (5× per week). Mice that were treated with 20 µg Val-boroPro and subsequently rejected MB49 were then rechallenged with MB49 (1×10⁶) or 76-9 rhabdomyosarcoma (5×10⁵) on day 56 post-primary challenge (n = 5/group). Saline control, initial treatment (closed squares), naïve mice (that did not previously reject tumors) injected with MB49 (closed triangles) or 76-9 (closed diamonds) at time of rechallenge, mice that previously rejected MB49 during Val-boroPro and rechallenged with MB49 (open circles) or 76-9 (inverted open triangles). (B) Mice were inoculated with MB49 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1A</a> and treated with Val-boroPro or saline (closed squares) (n = 5/group). CD4⁺ and/or CD8⁺ T cells were depleted with anti-CD4 and/or anti-CD8 monoclonal antibodies as outlined in methods. Tumor volumes in Val-boroPro treated mice without T cell depletion (closed squares) were significantly smaller than either CD4 (closed triangles) or CD8 (inverted open triangles) depleted groups (p<0.05) and CD4/CD8 depleted group (closed circles) (p<0.001) Open circles represent anti-CD4/anti-CD8 without Val-boroPro treatment. (C) Male (closed circles) and female (closed squares) mice were challenged subcutaneously with 10⁶ HY-expressing tumor MB49 on day 0 and were treated with 20 µg Val-boroPro 5×/week for two weeks (n = 5/group). P<0.01 for all timepoints beyond day 15. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g002" target="_blank">Figures 2A–C</a> are representative of 3 experiments.</p

Antitumor activity with Val-boroPro is associated with increased trafficking of DCs.

<p>(A–B) B6CD45.1 congenic mice were treated with 20 µg Val-boroPro for three days, and CD11c⁺ cells from spleens and LNs were magnetic bead purified and injected at a dose of 5×10⁶ into lateral tarsals of C57BL/6 (CD45.2⁺) recipients. Immediately following CD11c⁺ cell injection, recipients were treated with Val-boroPro or saline (n = 3/group). Popliteal lymph nodes were harvested 15 hours following Val-boroPro treatment for flow cytometric analysis. (A) Representative dot plots. (B) Bar graph showing statistically more CD45.1⁺ adoptive transferred DCs in the popliteal LN in recipients of DCs from Val-boroPro treated donors also receiving a single injection of Val-boroPro compared to all other group (*p<0.05, **p<0.01, Mann-Whitney). (C) Purified CD11c⁺ cells from Val-boroPro treated GFP⁺ mice were injected intratumorally into established MB49 tumors. Mice were treated immediately thereafter with one dose of Val-boroPro (n = 3/group). Tumor-draining inguinal lymph nodes were harvested 15 hours later and imaged by fluorescent microscopy. Representative lymph nodes from mice receiving CD11c⁺ cells from Val-boroPro treated donors and Val-boroPro post injection. Experiments displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g008" target="_blank">Figures 8A–C</a> were conducted 2 times.</p