The DNA methyltransferase inhibitor, guadecitabine, targets tumor-induced myelopoiesis and recovers T cell activity to slow tumor growth in combination with adoptive immunotherapy in a mouse model of breast cancer

Tumors avoid immune detection and attack through a variety of mechanisms that circumvent the anti-tumor response. DNA hypermethylation, though reversible, can silence the expression of immunogenic antigens; this makes the immune system less effective, especially during immunotherapeutic interventions [1]. Tumors also recruit regulatory immune cells, including MDSCs, which dampen the adaptive immune response. Patients with higher levels of circulating MDSCs have increased primary tumor growth [2], higher metastatic burden [3], more advanced clinical cancer stage [4, 5], and shorter overall survival [3, 6]. Based on these findings and multiple reports in mouse models that implicate MDSCs as key obstacles to successful cancer immunotherapy, there has been much interest in eliminating the suppressive nature of MDSCs to improve patient outcomes [7‐10].

The myeloid compartment in cancer has been extensively reviewed, especially MDSCs [11]. MDSCs are divided into two subsets, monocytic and granulocytic MDSCs. Both types of MDSCs have been shown to be suppressive in both murine tumor models and in several human cancers. Monocytic MDSCs generate nitric oxide as a mechanism of suppression, whereas granulocytic MDSCs express large amounts of reactive oxygen species and arginase-1 that results in suppression of T cell function [11]. MDSCs have been found in the spleen, blood, liver, and tumor of tumor-bearing animals. These suppressive cells have been found to accumulate in various types of murine tumor models and human cancer, from murine hepatic carcinoma models [12], breast cancer models [13], to human ovarian cancer [14], and many more.

Our lab has previously published on the effects of the DNA methyltransferase inhibitor (DNMTi), decitabine. Using the aggressive murine breast cancer line, 4 T1, we found that decitabine improved the immunogenicity of these cells in vitro, and augmented the effects of adoptive immunotherapy (AIT) in vivo [10]. While decitabine caused a reduction in tumor-induced MDSC accumulation, the underlying mechanism behind this was never investigated. In our current study, we have expanded upon these findings with the second-generation DNTMi, guadecitabine, and investigated its mechanism of action in tumor reduction. Like the active metabolite decitabine, we found that guadecitabine diminished tumor-induced granulocytosis in 4 T1 tumor-bearing mice. As a result of the reduced MDSC accumulation, guadecitabine rescued immune activation and was able to reduce tumor growth in a T cell-dependent manner. Guadecitabine was similarly effective in the E0771 model of murine breast carcinoma. Finally, we found that guadecitabine in combination with AIT resulted in prolonged survival in both 4 T1 and E0771 breast cancer models. Because of these advantageous effects, guadecitabine could prove to be a beneficial new drug to reduce systemic immune suppression and augment the effectiveness of immunotherapy in cancer patients.

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 [28, 32] and 5-Azacitadine (AZA) [33], as well as other chemotherapy drugs, including gemcitabine [34, 35], doxorubicin [8] and docetaxel [36]. Although MDSCs are not being directly targeted, they seem to be more susceptible to their effects as demonstrated in this study. 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 (Fig. 1c-e). Based on the time-course experiment (Fig. 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 (Fig. 2c, Supplemental Fig. 3e). This prevents increased MDSC circulation (Fig. 2d) and accumulation within the spleen (Fig. 2b). Surprisingly, we found that the similarly proliferative 4 T1 tumor cells were not vulnerable to cytotoxic effects of in vivo guadecitabine treatments (Fig. 3f).

Within the spleen of tumor-bearing mice we showed an accumulation of MDSCs in the red pulp (Supplemental Fig. 2d). 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, 37]. 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 used guadecitabine to ablate the suppressive splenic environment. We found that IFNγ production within the dLN is comparable between control and guadecitabine-treated mice (Fig. 4a). Upon recirculating through the spleen, however, CTLs from control tumor-bearing mice have diminished activation (Fig. 4b), even though the number of activated cells remained the same (Fig. 4c). These data support 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 (Fig. 2a), indicating guadecitabine may have a beneficial impact beyond the removal of regulatory myeloid populations from the spleen. The reduced suppressive activity in the spleen and tumor environments may be the reason for the reduction in tumor growth (Supplementary Fig. 7a,b). A recent study examining MDSC subsets in the peripheral blood of patients with multiple types of cancer found that arginase1 generated by granulocytic MDSCs was the main T cell inhibition mechanism [38]. In our study, guadecitabine was able to dramatically reverse the generation of arginase1, as well as dramatically reducing granulocytic MDSCs. Additionally, we demonstrated increased Ifng expression in the tumors of guadecitabine-treated mice (Fig. 4e). Kim et al. recently demonstrated across several murine models of triple negative breast cancer (TNBC) and in patient samples of TNBC that tumor infiltrating neutrophilic myeloid cells (TINs) were immunosuppressive and contribute to poor prognostic outcomes in patients [13]. In contrast, tumor infiltrating macrophages or monocytic myeloid cells (TIMs) were associated with increased responsiveness to checkpoint inhibitors in the murine models and better prognosis in humans [13]. Using guadecitabine treatment, Gr1⁺ cells are depleted, which include both MDSCs and neutrophils, but beneficial F4/80⁺ TIMs are not affected (Supplemental Fig. 7b). The enhanced tumor immunity may additionally 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 and costimulatory markers such as MHC II and CD80/86 (Fig. 1f, Supplemental Fig. 1b). Since myeloid populations have been previously shown to be extremely plastic [39], these data suggest that guadecitabine pushes suppressive MDSCs to develop into an immune-stimulatory phenotype that may 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 splenin environment, 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 tumors, combination therapy with AIT+guadecitabine compounded this effect and resulted in persistent tumor suppression (Fig. 5b) and prolonged survival (Fig. 5e). It is interesting to note that in the AIT experiments, guadecitabine was administered earlier at days 3, 4, 5, and 6 (Fig. 5), rather than days 10, 11, 12, and 13 (Figs. 1, 2, 3 and 4). This dosing schedule still resulted in slower tumor growth through day 16, although the reasons why are unclear. 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 Fig. 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.

Chimeric antigen receptor (CAR) T cells are genetically engineered to be specific for a designated target tumor antigen. These cells have been shown to be successful in the clinic against CD19⁺ B cell neoplasms. New CARs are being generated against antigens on solid tumors [40]. One of the biggest blocks to CAR treatment in solid tumors has been the suppressive environment created by MDSCs [40]. The addition of guadecitabine to CAR therapy may hold the key to favorably altering this environment and, as seen with the AIT experiments in this study, help augment the efficacy of adoptively transferred T cells.

Finally, we showed the efficacy of guadecitabine in slowing the growth of another tumor line on a different background strain. There were no observable strain differences noted relating to guadecitabine treatment between the C57Bl/6 J and Balb/cJ mice. The tumor line E0771 is not known to elicit a robust leukemoid reaction, but studies still indicate a suppressive role for MDSCs in this model [41, 42]. Overall, we observed a similar and persistent reduction in tumor growth with guadecitabine alone, or in combination with AIT.

In conclusion, we have shown that guadecitabine treatment is effective against the aggressive 4 T1 and E0771 breast cancer lines targets excessive myelopoiesis within the bone marrow and greatly reduces MDSCs within the spleen, tumor, and blood. This eliminates the systemic T cell suppression, thereby rescuing the host’s anti-tumor CTL response. We’ve shown that guadecitabine treatment significantly improves AIT treatment by providing an environment in which the transferred T cells can maintain their antigen-specific activity long-term. Because of these advantageous effects of guadecitabine on multiple targets, it should prove to be a beneficial new drug to reduce systemic immune suppression and augment the effectiveness of immunotherapy in cancer patients. The immunosuppression relief provided by guadecitabine may also enhance responsiveness to other cancer treatments such as checkpoint inhibitors or CAR T cells that are inhibited by suppressive myeloid cells.