There is great difficulty in treating neoplastic disease, especially when the tumor becomes resistant to chemotherapy. Today, the most promising interventions involve boosting the patient’s own immune system to detect and destroy abnormal cells. Immunotherapy has emerged as a highly promising and effective treatment for a variety of cancer types, but still only a minority of patients exhibit strong objective responses. Tumors can employ several “tactics” to avoid recognition and suppress anti-tumor immune response. Through hypermethylation, tumors can silence immunogenic antigens to avoid cell-mediated killing. Additionally, the tumor environment can induce massive myelopoiesis, causing suppressive MDSCs to accumulate in the bone marrow, spleen, circulation, and in the tumor. For these reasons, there is great interest in finding ways to reverse these effects, thus allowing the immune system to clear the tumor.
Several groups have reported on the myelo-depleting properties of demethylating drugs such as decitabine(20,24) and 5-Azacitadine (AZA)(25), as well as other chemotherapy drugs, including gemcitabine(26,27), doxorubicin(8) and docetaxel(28). Although MDSCs are not being directly targeted, they seem to uptake the drugs more readily and are more susceptible to their effects. Guadecitabine, also known as SGI–110, was specifically designed to be resistant to degradation by cytidine deaminase and prolong the exposure of tumor cells to the active metabolite, decitabine. In vivo, guadecitabine treatments resulted in a near-complete absence of MDSCs in tumor-bearing mice (Figure 1c-e).. Based on the time-course experiment (Figure 2),, it appears that guadecitabine treatment is preventing, rather than reversing, MDSC accumulation. We believe guadecitabine targets the bone marrow by diminishing the highly proliferative myeloid progenitors (Figure 2c).. This prevents increased MDSC circulation (Figure 2d) and accumulation within the spleen (Figure 2b).. Surprisingly, we found that the similarly proliferative 4T1 tumor cells were not vulnerable to cytotoxic effects of in vivo guadecitabine treatments (Figure 3f)..
Within the spleen of tumor-bearing mice we showed an accumulation of MDSCs in the red pulp (Supplemental Figure 1d).. This perilymphoid localization puts the MDSCs in contact with recirculating CD8+ CTL. Several tumor studies have portrayed the spleen as an inhibitory environment that can diminish CTL function(7,29). In experiments by Ugel et al, the investigators removed the inhibitory MDSC environment through splenectomies(7). Although this did not affect tumor size, they found that T cell activation was recovered despite normal MDSC frequency within the blood and other tissues. This highlights the spleen’s unique role as an isolated region of suppression with the ability to severely dampen the anti-tumor immune response. In the present study, we use guadecitabine to ablate the suppressive splenic environment. We found that IFN production within the dLN is comparable between WT and guadecitabine-treated mice (Figure 4a).. Upon recirculating through the spleen, however, WT CTLs have diminished activation (Figure 4b) even though the number of activated cells remained the same (Figure 4c).. This data supports the role of the spleen as an important suppressive zone that contributes to tumor progression.
Unlike Ugel’s splenectomy experiments, our treatment additionally resulted in slower tumor growth (Figure 2a),, indicating guadecitabine may have a beneficial impact beyond the removal of regulatory myeloid populations. The enhanced tumor immunity may arise from guadecitabine’s effect on MDSC phenotype. Although the majority of the MDSCs are eliminated, a small percentage of cells remained that are induced to express APC markers such as MHC II and CD80/86 (Figure 1f).. These data suggest that guadecitabine pushes suppressive MDSCs to develop into an immune-stimulatory phenotype to augment immune activation within the spleen.
The Ugel experiment also emphasizes a significant problem with a popular and promising clinical therapy. Animals that underwent sham surgeries responded poorly to AIT compared to those that received splenectomies. When the antigen-experienced T cells circulate through the suppressive spleen, they are inactivated despite being primed to target the tumor. Here we have shown a similar phenomenon; while AIT was effective in slowing the growth rate of the tumor, combination therapy with AIT+guadecitabine compounded this effect and resulted in persistent tumor suppression (Figure 5b) and prolonged survival (Figure 5e).. It is interesting to note that in the AIT experiments, guadecitabine was administered earlier at days 3, 4, 5, and 6 (Figure 5),, rather than days 10, 11, 12, and 13 (Figures 1–4).. This dosing schedule still resulted in slower tumor growth through day 16, although the reasons why are unclear. As we showed in Figure 2b, splenomegaly does not occur in our model until around day 14, correlating with the accumulation of MDSCs. We hypothesize that guadecitabine treatment targets the bone marrow, disrupting the abnormal myelopoiesis before it can begin. Further, we tested the effectiveness of delaying adoptive T cell transfer until the final guadecitabine treatment rather than being delivered concomitantly with the initial treatment (Supplemental Figure 7).. The delayed AIT alone was more effective at days 6 and 7 than at days 3 and 4, perhaps because the tumor has become more immunogenic and vascularized by the later date.
Finally, we showed the effectiveness of guadecitabine in slowing the growth of another tumor line on a different background strain. Although E0771 is not known to elicit a robust leukemoid reaction, studies still indicate a suppressive role for MDSCs in this model(30,31). We observed a similar and persistent reduction in tumor growth with guadecitabine alone, or in combination with AIT.