Elucidation of the molecular mechanisms of action of anti-cancer compounds reveals possible combination strategies for anti-cancer therapy

Abstract: Cancer develops due to accumulation of mutations or epigenetic changes within normal cells, usually over a considerable period of time. When cancer patients are diagnosed at late stage, the tumor cells are often more heterogenous and aggressive, which results in tumor resistance to anti-cancer therapies and therefore can be lethal. In order to overcome drug resistance, effective combinational therapies are needed. My PhD project aimed to explore the mechanisms of action of anti-cancer compounds, including p53 activating compounds, chemotherapeutic drugs and hormonal therapy. We also aimed to discover the most efficient combination therapies based on the mechanisms of action we uncovered which potentially can guide clinical practice. In paper I, we explored how pharmacological activation of wtp53 by MDM2 inhibitors promotes immune response within tumor cells and TME. We found that p53 activating compounds or peptide, including Nutlin-3a, AMG-232 and ATSP-7041, de-repressed retrotransposons, such as ERVs and Long interspersed nuclear element-1 (LINE-1), via repression of DNA methyltransferase 1 (DNMT1) and Lysine-specific histone demethylase 1A (LSD1) encoded by lysine demethylase 1A (KDM1A). De-repression of ERVs triggered IFN response followed by activation of downstream signaling involving antigen processing and presentation (APP) which enhanced exogenous neoantigen ovalbumin (OVA) presentation on the surface membrane of cancer cells. We also confirmed that after p53 activation there was an induction of IFN response in both mouse model and tumor samples from patients engaged in MDM2 inhibitor ALRN-6924 clinical trial. We observed that p53 activating compounds upregulated the expression of APP genes and PD-L1 in B16 murine melanoma. Therefore, we investigated the effect of combination of p53 activating peptide ATSP-7041 or its advanced analogue ALRN-6924 with anti-PD-1 antibody in two different tumor bearing mouse models. We found that the ATSP-7041 overcame tumor resistance to anti-PD-1 therapy and significantly decreased the tumor growth rate in comparison with control or single agent treated mice. Moreover, p53 activating peptide facilitated the infiltration of anti-tumor immune cells, consisting of CD4+ T cells, CD8+ T cells and B cells and decreased the immune suppressive myeloid-derived suppressor cells (MDSCs) within TME of B16 tumor bearing mouse model. Our results reveal a novel finding that p53 activating compounds can de-repress ERVs via suppression of two different chromatin modifiers, induce IFN response in cancer cells, mouse model and cancer samples from patients, and overcome immunologically cold tumor’s resistance to immune checkpoint blockade in mouse models. These findings are very promising to translate to clinical applications. In paper II, we investigated the mechanisms of breast cancer resistance to TMX and how we could overcome the resistance by three anti-cancer compounds. We found a higher SULT1A1 expression in spontaneous TMX resistant breast cancer cell line MCF7-TMXR compared to normal ER+ MCF7 breast cancer cell line. The depletion of SULT1A1 in MCF7-TMXR sensitized cells to TMX treatment again. Furthermore, TMX treatment induced SULT1A1 expression in both patient-derived samples and in ER+ MCF7 breast cancer cell line. We found that anti-cancer activities of RITA, AF and ONC-1 were dependent on SULT1A. The mechanism of action of these compounds, including induction of ROS as a result of inhibition of thioredoxin reductase 1 (TrxR1) activity and DNA damage, was also SULT1A1-dependent. We further showed that these three anti-cancer compounds had synergistic effect with TMX in ER+ MCF7 breast cancer cell line. In line with these results, MCF7-TMXR had better response to RITA and AF than normal ER+ MCF7. We observed that the pretreatment with TMX of patient breast cancer samples, or TMX unresponsive breast cancer samples sensitized to RITA treatment. Our data suggest that the induction of SULT1A1 is responsible for breast cancer resistance to TMX treatment in cancer cell lines and patient tumor samples. Resistance to TMX can be overcame by combination with anti-cancer compounds bioactivated by SULT1A1, which can be potentially translated into clinical setting to improve the clinical outcome. In paper III, we explored the mechanisms of mtp53-mediated evasion of cancers from the immune response. We addressed the question whether Apr-246, originally discovered as mtp53 activating compound, can recover the anti-tumor immune surveillance in cancers carrying mtp53. By analyzing TCGA data, we found that breast cancer patients carrying mtp53 display chronic inflammation characterized by elevated TIS score and IFN signaling, in comparison to breast cancer patients carrying wtp53. We found that mtp53 gained the function to upregulate IFNs and ERVs, a well-established upstream stimulator of IFN signaling. Moreover, we found that the majority of genes comprising a 26 gene signature that defines activated CD8+ T cell were upregulated in breast cancer patients carrying mtp53. Cytokines involved in CD8+ T cells attraction, proliferation and activity were upregulated, while Transforming growth factor beta (TGF-β) that inhibits T cell function was downregulated in breast cancers carrying mtp53. These data imply that breast cancers carrying mtp53 must develop mechanisms to escape from immune surveillance. Next we detected an enhanced expression of several ICs in samples from breast cancer patients carrying mtp53. Elevated expression of ICs is a well-studied tumor intrinsic mechanism to suppress immune response. We found that mtp53 acquired novel gain-of-function (GOF) to upregulate ICs in a set of cancer cell lines. Moreover forkhead box P3 (Foxp3), a well- established Treg cell marker, was also increased within TME of breast cancer patients carrying mtp53, indicating a tumor extrinsic mechanism to evade from CD8+ T cell surveillance. We further found that Apr-246 could induce acute IFN or repress ICs in a panel of cancer cell lines, while promoted CD4+ T cells infiltration and prevented CD8+ T cells exhaustion within the TME of a mtp53 tumor bearing mouse model. Together, our data suggest that cancer cells carrying mtp53 can escape immune surveillance die to enhanced inflammatory signaling, together with upregulated ICs. Apr-246 could recover the immune surveillance by inducing acute IFN activation or repressing ICs expression in cancer cell lines, and promoting anti-tumor immune response in in vivo mouse model.