Immune checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TME’s such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs) whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti–PD-1, anti–CTLA-4, or both, significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic-MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B–producing CD8+ T effector cells in mice treated with combination therapy. Gene expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

In cancers with tumor infiltrating lymphocytes (TIL), monoclonal antibodies (mAbs) that block immune checkpoints such as CTLA-4 and PD-1/PD-L1 promote antitumor T cell immunity. Unfortunately most cancers fail to respond to single agent immunotherapies. T regulatory cells, myeloid derived suppressor cells (MDSCs), and extensive stromal networks within the tumor microenvironment (TME) dampen antitumor immune responses by preventing T-cell infiltration and/or activation. Few studies have explored combinations of immune checkpoint antibodies that target multiple suppressive cell populations within the TME, and fewer have studied the combinations of both agonist and antagonist mAbs on changes within the TME. Here we test the hypothesis that combining a T cell-inducing vaccine with both a PD-1 antagonist and CD40 agonist mAbs (triple therapy) will induce T cell priming and TIL activation in mouse models of non-immunogenic solid malignancies. In an orthotopic breast cancer model and both subcutaneous and metastatic pancreatic cancer mouse models, only triple therapy was able to eradicate most tumors. The survival benefit was accompanied by significant tumor infiltration of IFNγ-, Granzyme B-, and TNFα-secreting effector T cells. Further characterization of immune populations was carried out by high dimensional flow cytometric clustering analysis and visualized by t-distributed stochastic neighbor embedding (t-SNE). Triple therapy also resulted in increased infiltration of dendritic cells, maturation of antigen presenting cells, and a significant decrease in granulocytic MDSCs. These studies reveal that combination CD40 agonist and PD-1 antagonist mAbs reprogram immune resistant tumors in favor of antitumor immunity.

Key Points• ATRA and FLT3 TKIs have synergistic activity against FLT3/ITD 1 AML cell lines and patient samples.• Combination reduces the leukemia stem cell population and improves survival in genetic and xenograft AML mouse models.

FLT3 tyrosine kinase inhibitors (TKI) have been tested extensively to limited benefit in acute myeloid leukemia. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to TKI treatment that are associated with an incomplete response. Here we identified reactivation of ERK signaling within hours following treatment of FLT3/ITD AML cells with selective inhibitors of FLT3. When these cells were treated with inhibitors of both FLT3 and MEK in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared to either drug alone. ERK reactivation was also observed following inhibition of other tyrosine kinase-driven cancer cells, including EGFR-mutant lung cancer, HER2-amplified breast cancer and BCR-ABL leukemia. These studies reveal an adaptive feedback mechanism in tyrosine kinase-driven cancers associated with reactivation of ERK signaling in response to targeted inhibition.

FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately one third of acute myeloid leukemia cases. The most common FLT3 mutations in acute myeloid leukemia are internal tandem duplication (ITD) mutations in the juxtamembrane domain (23%) and point mutations in the tyrosine kinase domain (10%). The mutation substituting the aspartic acid at position 838 (equivalent to the human aspartic acid residue at position 835) with a tyrosine (referred to as FLT3/D835Y hereafter) is the most frequent kinase domain mutation, converting aspartic acid to tyrosine. Although both of these mutations constitutively activate FLT3, patients with an ITD mutation have a significantly poorer prognosis. To elucidate the mechanisms behind this prognostic difference, we have generated a knock-in mouse model with a D838Y point mutation in FLT3 that corresponds to the FLT3/D835Y mutation described in humans. Compared with FLT3/ITD knock-in mice, the FLT3/D835Y knock-in mice survive significantly longer. The majority of these mice develop myeloproliferative neoplasms with a less-aggressive phenotype. In addition, FLT3/D835Y mice have distinct hematopoietic development patterns. Unlike the tremendous depletion of the hematopoietic stem cell compartment we have observed in FLT3/ITD mice, FLT3/D835Y mutant mice are not depleted in hematopoietic stem cells. Further comparisons of these FLT3/ D835Y knock-in mice with FLT3/ITD mice should provide an ideal platform for dissecting the molecular mechanisms that underlie the prognostic differences between the two different types of FLT3 mutations.

There have been a number of clinical trials testing the efficacy of FLT3 tyrosine kinase inhibitors (TKIs) in acute myeloid leukemia (AML). patients harboring a constitutively activating mutation in FLT3 However, there has been limited efficacy, most often due to inadequate achievement of FLT3 inhibition through a variety of mechanisms In a previous study, TTT-3002 was identified as a novel FLT3 inhibitor with the most potent activity to date against FLT3 internal tandem duplication (FLT3/ITD) mutations Here the activity of TTT-3002 is demonstrated against a broad spectrum of FLT3 activating point mutations (FLT3/PMs), including the most frequently occurring D835 mutations The compound is also active against a number of point mutations selected for in FLT3/ITD alleles that confer resistance to other TKIs, including the F691L gatekeeper mutation TTT-3002 maintains activity against relapsed AML patient samples that are resistant to sorafenib and AC220 Studies utilizing human plasma samples from healthy donors and AML patients indicate that TTT-3002 is only moderately protein bound compared to several other TKIs currently in clinical trials Tumor burden of mice in a FLT3 TKI-resistant transplant model is significantly improved by oral dosing of TTT-3002 Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKIs in the treatment of FLT3-mutant AML

All-trans retinoic acid (ATRA) has been very successful in the subtype of acute myelogenous leukemia known as acute promyelocytic leukemia due to targeted reactivation of retinoic acid signaling. There has been great interest in applying this form of differentiation therapy to other cancers, and numerous clinical trials have been initiated. However, ATRA as monotherapy has thus far shown little benefit in nonacute promyelocytic leukemia acute myelogenous leukemia. Here, we review the literature on the use of ATRA in combination with chemotherapy, epigenetic modifying agents and targeted therapy, highlighting specific patient populations where the addition of ATRA to existing therapies may provide benefit. Furthermore, we discuss the impact of recent whole genome sequencing efforts in leading the design of rational combinatorial approaches.

While novel FLT3 tyrosine kinase inhibitors (TKIs) are increasingly efficient, when used as monotherapy they achieve only limited clinical responses in patients with FLT3/ITD acute myeloid leukemia (AML). Leukemia stem cells (LSCs) that share characteristics with normal hematopoietic stem cells have been implicated in persistence of minimal residual disease (MRD) and resistance to chemotherapy. Elimination of LSCs is paramount for any curative therapy in AML. Retinoic acid (RA) pathways are essential for normal and malignant stem cell homeostasis (Ghiaur G et al. 2013, Su M et al. 2015). We and others have previously reported that combining all-trans RA (ATRA) with FLT3 inhibitors leads to synergistic FLT3/ITD+ cell killing (Ma H et al. 2013, Chi et al. 2015). However, bone marrow (BM) mesenchymal cells were shown to inactivate ATRA via expression of CYP26 and thus protect LSCs from pro-differentiation effects. Here we investigated whether inhibition of CYP26 via talarozole and/or use of the CYP-resistant retinoid tamibarotene can overcome stromal protection and restore the full cytotoxic effect of the TKIs against FLT3/ITD+ LSCs in the stem cell niche. The combinatorial effect of FLT3 TKIs, retinoids, and talarozole was assessed in FLT3/ITD+ AML patient samples and cell lines in liquid as well as stroma co-culture. Our studies reveal synergistic activity against FLT3/ITD+ cells between FLT3 TKIs and ATRA in liquid culture, with combination index (CI) values of 0.1-0.7, leading to a significant induction of apoptosis. This treatment was rendered inefficient by co-culture in the presence of BM stroma (1.7- to 3-fold reduction in AnnexinV+ cells, P < 0.001). Inhibition of stromal CYP26 via talarozole or by-passing stromal CYP26 via a CYP26 resistant retinoid restored the TKI's ability to induce apoptosis of FLT3/ITD+ cell lines and patient samples cultured on stroma to a similar level as achieved with a FLT3 inhibitor alone in liquid culture. Colony-forming unit (CFU) assays further demonstrated decreased clonogenicity of FLT3/ITD+ cells on stroma co-culture upon treatment with TKI, ATRA, and talarozole (85% reduction in CFUs vs. 57% without talarozole, P < 0.001) or FLT3 TKI plus tamibarotene (86% reduction vs. 50% with ATRA, P < 0.01). Similar to treatment with CYP26 inhibitor, genetic deletion of CYP26 in the BM stroma also partially rescued the combinatorial effect between ATRA and TKIs otherwise lost in stroma co-culture conditions. Using various mouse models of FLT3/ITD leukemia (Molm14 and patient sample xenografts, transgenic FLT3/ITD;NUP98HOXD13), we have observed that as with AML patients, treatment with FLT3 TKIs alone is not sufficient to eliminate MRD and cure the disease. Addition of ATRA to the in vivo treatment with sorafenib (a FDA approved TKI with activity against FLT3) greatly decreased the level of engraftment of leukemia cells in mice, and significantly increased median survival compared to either drug alone. Furthermore, there was significant depletion of the LSCs as measured by limiting dilution transplantation of BM from mice treated with sorafenib and ATRA in combination. Nevertheless, elimination of the last bastion of LSCs remained elusive even after combination therapy with ATRA and sorafenib in these mice. Our studies suggest that this effect may be due to the enhanced metabolism of retinoids in the BM, thereby diminishing the combinatorial effect of retinoids and FLT3 TKIs. Therefore, the use of CYP-resistant retinoids or CYP inhibitors in combination with TKIs may improve the cure rate of FLT3-mutant AML in mouse models. We propose the existence of RA-low microenvironments in the BM where the combinatorial activity with TKIs against FLT3/ITD is impaired and thus, persistence of MRD is possible. Our findings support the development of a clinical trial of FLT3 TKIs, retinoids, and/or CYP inhibition in relapsed/refractory FLT3-mutant AML patients. Disclosures Aplan: NIH Office of Technology Transfer: Patents & Royalties.