Importance of IC pathways in immune evasion of AML as well as their blockade with specific agents in AML treatment has been underlined in several experimental studies which involved AML cell lines and murine models.

Constitutive expression of regulatory cell surface antigen CTLA-4 expression in more than 80% of AML samples was first reported two decades ago (33) and in 2006, its’ blockade with monoclonal antibodies were reported to enhance T cell responses in AML in-vitro (34). In a study involving a DA1-3b mouse model of AML, leukemic cells were reported to be present months despite the presence of an effective antileukemic immune response. Persistent leukemic cells were reported to have enhanced B7-H1 (PD-L1) and B7.1 expressions and resistant to cytotoxic T cell (CTL) mediated killing (35). The authors stated that an effective immunotherapeutic intervention should facilitate leukemia rejection and targeting overcoming the mechanisms that lie behind tumor dormancy and revealed that inhibition of B7-H1 (PD-L1) and B7.1/CTLA-4 interactions augmented CTL-mediated killing of the persistent cells as well as prolonging survival of naive mice injected with persistent leukemic cells. However, it should be noted that targeting B7.1/CTLA-4 and PD-1/PD-L1 axes may target different mechanisms compared to monotherapies (36), and elucidating such pathways in leukemias may pave the way for novel combinatorial therapies.

In terms of PD-1/PD-L1 axis, numerous experiments revealed upregulated expressions of both proteins in murine leukemia cells while demonstrating that genetic ablation or pharmacological inhibition of PD-1 can suppress leukemic cell proliferation and enhance survival in AML bearing mice (37). Combinatorial administration of innate immune agonists along with an ICI has revealed promising results by enhancing anti-tumor activity in a preclinical AML model: an innate immune agonist 5,6-dimethylxanthenone-4-acetic acid (DMXAA) activated the stimulator of interferon genes (STING) pathway that promoted dendritic cell maturation and in turn, maturation of leukemia-specific T-cells, resulting in a prolonged overall survival in leukemic mice (38). In anti-tumor responses, type I interferons (IFN) promotes the infiltration of CD8+ T cells, hence acts as a bridge between the innate and adaptive immunity (39, 40). This pathway also activates STAT6 and nuclear factor kappa B (NF-κB) pathways that result in the production of inflammatory mediators including TNF-α, IL-6 and CCL2/MCP-1 (41, 42). Unlike solid tumors, type I IFN response is shown not to be activated in hematological malignancies and activating STING pathway to promote anti-leukemic T cell responses stands out as a promising strategy (43). However, expression of immunosuppressive indolamine-2,3-dioxygenase (IDO) and upregulation of PD-L1 as a response to IFN-γ may be the restricting factors for the administration of STING agonists as a single agent in AML treatment. Thus, determination and inhibition of immune escape pathways induced by STING activation may enable STING agonists’ administration in the clinical setting. In line with this hypothesis, DMXAA inhibited the growth of murine AML cell line C1498 and increased PD-L1 expression while combination of PD-1 inhibition along with DMXAA therapy boosted activated host T cell numbers and bone marrow PD-1/L1/L2 expression, reducing disease burden and prolonging overall survival in-vivo (44). In an in-vitro study, DMXAA exposure promoted PD-L1 expression while leading to a slight increase in apoptosis and IL-6 and IFN-b production in C1498 AML cell line while coupling DMXAA with an anti-PD-1 antibody significantly reduced disease burden and extended general survival in C1498 grafted leukemic mice (45). Mice receiving combinatorial treatment exhibited boosted memory T-cells and mature dendritic cells along with lesser numbers of regulatory T-cells, proving apoptosis of leukemic cells. These findings were further supported by increased serum levels of type I interferons (IFN) and IFN-γ. These studies suggest that STING agonists can be used in combination with ICI for enhanced anti-tumor efficacy. Besides DMXAA, other STING agonists include GSK3745417 that has been shown promising anti-cancer activity on AML cell lines as well as primary AML cell cultures and MIW815 (ADU-S100) which recently have been reported to induce systemic immune activation while being well tolerated in patients with advanced/metastatic cancers, though AML was not investigated in the latter (46, 47). A recent study revealed that a novel STING agonist SHR1032 enhanced anti-tumor immunity and induced AML apoptosis under in-vitro and in-vivo settings (48). Besides AML, STING agonists have been under evaluation for the treatment of other solid and hematological malignancies: Ulevostinag (MK-1454) has been tested in combination with pembrolizumab in participants with advanced/metastatic solid tumors or lymphomas (49), while GSK3745417 is currently being tested either alone or in combination with PD-1 inhibitor dostarlimab (50), and BMS-98630 is being tested alone or in combination with nivolumab and ipilimumab in patients with advanced solid tumors (51). However, there are certain questions to be addressed before the implementation of STING agonists in the field of immune oncology, including whether the overstimulation of the pathway can induce autoimmune conditions, or if the pathway is a valid target in case of epigenetic silencing of STING (52).

Recent findings suggest that AML cells express high levels of TIM-3 and release galectin-9 (Gal-9) that impair activity of cytotoxic T cells and NK cells (53). The association between PI3K/Akt/mTOR signaling pathways and the regulation of immune checkpoint ligands including PD-L1, Galectin-9 (Gal-9), and CD155 was investigated in human AML cell line HL-60 in-vitro. For this purpose, cells were treated with idelalisib as PI3K inhibitor, MK-2206 as Akt inhibitor, and everolimus as mTOR inhibitor either in a single or combined format (54). Combinatorial treatment of HL-60 cells with two or three inhibitors diminished the expression levels of PD-L1, Gal-9, and CD155 checkpoint ligands, decreased proliferation and enhanced apoptosis. This study revealed that besides their cytotoxic properties, drugs targeting the PI3K/Akt/mTOR pathway play role in the regulation of ICP expression and interfere with immune evasion mechanisms of AML cells.

Recently, Xu et al. reported co-expression of PD-1 along with TIGIT on CD8+ T cells of AML patients’ bone marrow samples, moreover PD-1 and TIGIT positivity on CD8+ T cells showed positive correlation with age, suggesting greater T cell dysfunction in elderly patients. This study also revealed the increased frequency of PD-1+ and TIGIT+ CD8+ T cells in bone marrow samples compared to peripheral blood, a finding that indicates the importance of targeting immunosuppressive bone marrow microenvironment (BMM) in AML treatment (55). In another study aiming to characterize NK cell subsets of AML patients in bone marrow and peripheral blood, Brauneck et al. revealed TIGIT and poliovirus receptor-related immunoglobulin domain-containing (PVRIG) co-expression on NK cells of AML patients, and their simultaneous blockade enhanced the NK cell mediated killing in-vitro (56). In another study, Li et al. reported PVRIG ligand (poliovirus receptor-related 2, PVRL2) on AML patient blasts, and proven that blocking the PVRIG/PVRL2 axis enhanced NK cell activation and in turn, promoted killing of patient derived primary AML blasts (57).

CD47 is a macrophage ICP that is particularly involved in myeloid malignancies and has been identified as a leukemic stem cell marker in AML. Blockade of CD47-SIRPα pathway has been shown to increase several therapeutics in pre-clinical studies (58). Similarly, CD200 plays role in the formation of T regulatory cells (Tregs) is commonly overexpressed in AML blasts and shown to be associated with poor outcome (59). Along with CD200 on AML blasts, TIM-3 expression on peripheral blood T cells was proven to be involved in AML development, and these proteins hold the potential to serve as prognostic markers (60).

Programmed Death-1 Homolog (V-domain Ig suppressor of T cell activation, VISTA) is a novel co-inhibitory molecule that promotes immune evasion in solid tumors, and an in-vivo study revealed the connection between PD-1H and epigenetic modifications as well as their role in immune evasion in AML where DNA methyl transferase inhibition by 5-aza-2’-deoxycytidine (Decitabine) increased T cell infiltration that potentiated the anti-leukemic effect of the PD-1H blockade and significantly prolonged survival (61). VISTA has also been shown to be expressed on myeloid-derived suppressor cells (MDSCs) present in the peripheral blood of AML patients and contribute to the inhibition of T cell responses in AML (62). Moreover, authors reported a positive correlation between VISTA expression on MDSCs and PD-1 expression on T cells of AML patients, highlighting the potential of combinatorial VISTA and PD-1 inhibition in leukemia treatment. In an in-vitro study, both CTLA-4 and LAG-3 expression levels were reported higher in comparison with healthy controls in AML, and the receiver-operating characteristic (ROC) curve analysis suggested that CTLA-4 and LAG-3 co-positivity can be used as a diagnostic criteria for the disease (63).

It should be noted that even if the ICIs are promising in the cancer treatment, the broadly distributed immune-related adverse events (irAEs) may not be tolerable in some cases. To overcome this, some experimental studies focus on restricted immune checkpoint blockade such as α-PD-1 × α-CD3 × α-CD33, a bifunctional checkpoint inhibitory T cell–engaging antibody (CiTE) that directs T-cells to CD33 on AML cells with locally restricted immune checkpoint blockade (64). By the synergistic effect of ICI and avidity-dependent binding, PD-1 attachment improved T-cell activation (3.3-fold elevation of IFN-γ) and led to efficient and highly selective cytotoxicity against CD33+ PD-L1+ cell lines as well as patient-derived AML cells. In a murine xenograft model, CiTE induced complete AML eradication without initial signs of irAEs.

HMAs have been approved by FDA, and they are being used as epigenetic modifiers for the treatment of myelodysplastic syndromes (MDS) and acute AML patients, who are not eligible for induction chemotherapy (65, 66). It is reported that in these patients who underwent the treatment with HMAs, surface expression of ICPs (PD-L1, PD-L2, PD-1, and to a lesser extent, CTLA-4) increased in a dose-dependent matter. For the patients for whom the up-regulation of PD-L1 was to the greatest extent, it is reported that the response to HMA therapy was the shortest, and it was associated with a lower survival (67). Concerning these observations, clinicians suggested that HMA therapies lead to immune checkpoint activation and up-regulation, indicating that this resistance may be overcome by combining HMA with ICIs (68, 69).

In a phase 2 clinical trial, nivolumab was administered with azacitidine to a high-risk population of relapsed or refractory (R/R) AML patients. Among 70 patients, the response rate to therapy was 33%, with 22% being in complete remission or incomplete hematologic recovery. Grade 3/4 irAEs were reported in 11% of patients, the most frequent one being pneumonitis. For all 70 patients, the median survival was 6.3 months, while for 32 salvage-1 patients (the first therapy administered after all standard treatments proved ineffective), it was 10.5 months. This finding indicates a promising response rate for the combination therapy, as also stated by the authors. A greater response rate was recorded in patients with higher CD3+CD8+ T cell infiltration pre-therapy. Thus, it was reported that pre-therapy T cell infiltration can be considered an inflamed tumor marker and a biomarker that can be used in deciding which patient group would benefit from ICI-based treatments (70). In an expanded cohort study as a follow-up study to the clinical trial, the anti-CTLA-4 antibody ipilimumab was added to azacitidine and nivolumab regimen and administered to 24 R/R AML patients. The study has reported a 1-year overall survival of 45% in R/R AML patients. When this new triple combination treatment is compared in the aspect of the median overall survival, with the previous azacytidine and nivolumab double treatment and with the current treatment with hypomethylating agents, the results were respectively 10.5, 6.4 and 4.6 months. These findings demonstrate an encouraging and promising efficacy. Although regarding its safety, it is worth mentioning that in 6 patients (25%), grade 3/4 immune-related toxicity, including rash, pneumonitis, and colitis was reported (71). In another phase 2 study, the anti-PD-1 antibody pembrolizumab was administered to recently diagnosed R/R AML patients in combination with azacitidine. In this cohort, out of 29 eligible patients, 4 (14%) achieved complete remission or incomplete hematologic recovery, while 1 patient (4%) had partial remission. The median overall survival was 10.8 months. After 22 newly diagnosed older AML patients not eligible for intensive chemotherapy joined the study, out of 17 of whom were evaluable, 47% achieved complete remission or incomplete hematologic recovery, while 12% had partial remission. The new median survival was 13.1 months. These two cohorts display that azacitidine and pembrolizumab combination therapy proved beneficial in both R/R and recently diagnosed older patient groups. Grade 3/4 irAEs were observed in both patient groups, the ratios being more frequent (24%) in the first cohort and less (14%) in the second. Although at this point this treatment combination looks more suitable to newly diagnosed older patient groups, more specifically directed research is still needed (72).

A phase 1b/2 study reported that azacytidine leads to PD-1 and PD-L1 upregulation in AML which causes drug resistance that may be overcome by including the PD-1 inhibitor nivolumab. In a study, azacitidine was combined with nivolumab and administered to 35 relapsed AML patients. Out of 35 patients evaluated, the preliminary data from this study showed that 6 (18%) were in complete remission (CR) or CR with incomplete count recovery (CRi) and 5 (15%) were in hematologic improvement. A decrease in blast count greater than 50% was observed in 14 patients (26%), and the median overall survival was reported as 9.3 months (range, 1.8 - 14.3). Patients with CR/CRi, higher levels of pre-treatment CD3+ and CD8+T-cell infiltration were detected in bone marrow aspirates (73). In conclusion, azacytidine in combination with nivolumab yielded a promising and durable response in relapsed AML, and irAEs may be managed with systemic steroid administration.

In another multi-centered, randomized, international phase 2 clinical trial, azacitidine was administered to high-risk MDS or AML patients in combination with the anti-PD-L1 antibody durvalumab or as a single agent. In this study with 129 AML patients older than 65 years old who were not eligible for chemotherapy; a comparison between the azacitidine and Durvalumab combination therapy and azacitidine as a single agent therapy showed no statistically significant difference in total response rate (31.3% vs. 35.4%) or complete remission rate (17.2% vs. 21.5%). The overall survival rate was 13.0 and 14.4 months, respectively, with no unexpected side effects. Although this study portrays an important role regarding its comparatively larger sample size, it is worth considering that more than half of the patients did not continue with the study regimens, which might be taken into account in interpreting the results (74).

The resistance mechanisms and biomarkers playing role in processes that play role in treatment response are not yet fully explained, but a study from Herbrich et al. puts forth a possible explanation. In their study, Herbrich et al. evaluated the bone marrow and peripheral blood samples taken from nine relapsed or refractory AML patients who received azacitidine and anti-PD-L1 antibody avelumab using single-cell mass cytometry. Out of nine evaluable patients, four had an initial decrease in blast count, and seven showed a fast progression subsequently. Authors reported that in AML bone marrow, CD4+ and CD8+ T cells had a significantly lower proportion of naïve T cells at baseline, along with a smaller ratio of terminally differentiated CD8+ cells. Contrarily, the largest portion of T-cells in AML bone marrow consisted of the effector memory CD4 and CD8 cells. In these patients, a high PD-L2 protein expression was observed in AML cells, and PD-L1 expression was low in the samples taken at both baseline and during therapy. PD-L2 was also frequently expressed in the newly formed clones which were not present at baseline. These findings may indicate a possible explanation for the different response rates to PD-1 and PD-L1 inhibition observed during AML treatment. These findings also indicate that the immune cell distribution is significantly affected in AML patients’ bone marrow. The T cell distribution ratio and the different checkpoints that are expressed on AML cells, such as PD-L2, may pose a key in the consideration of the approach and response of the treatment (75, 76).

In their study, Berger et al. administered anti-PD-1 monoclonal antibody CT-011 (pidilizumab) to patients with advanced hematologic malignities in a phase 1 clinical trial, where pidilizumab was administered to 17 patients (8 being diagnosed with AML) in doses between 0.2 and 0.6 mg/kg. Complete remission was observed in one patient, while clinical benefit is reported in 33%. Although serious adverse events were reported in 4 patients, who were all diagnosed with AML and passed away later, the study reported that none of these were related to the treatment but to fulminate-resistant leukemia and that the dose aforementioned can be considered safe (77, 78). Currently, pidilizumab is also being investigated in combination with a dendritic cell vaccine on AML patients in complete remission (79). In a phase 1/1b multi-centered study performed with hematologic cancer patients who were in relapse following post-allogenic HSCT, the anti-CTLA-4 antibody ipilimumab was administered to the patients. In 22 patients who were receiving 10 mg/kg, four were diagnosed with extramedullary AML, and one was diagnosed with MDS which progressed to AML; five patients (23%) were in complete remission, 2 (9%) showed partial response and 6 (27%) had a decreased tumor burden. A sustained response for longer than a year was reported in four patients. Although this study was noted to be attainable in patients with AML post-allogenic HSCT; the irAEs were reported in 6 patients (21%) including one death reported. Graft versus host disease (GvHD) is also reported in 4 (14%), which resulted in the conclusion of further application of ipilimumab. Altogether, these data revealed promising results regarding ipilimumab administration in patients with post-allogenic HSCT relapsed AML (65, 80). In a phase 2 study evaluating the effect of PD-1 inhibition after cytotoxic chemotherapy on clinical response, 37 patients diagnosed with relapsed or refractory AML were administered high-dose cytarabine followed by IV 200 mg pembrolizumab on the 14^th day. The patients who responded to the treatment continued to receive pembrolizumab for two years. The overall response rate was 46%, the composite complete remission was 38%, and the median overall survival was 11.1 months. For refractory or early relapsed patients, and for patients who received the treatment as the first salvage, the median overall survival was 13.2 and 11.3 months, respectively, which was considered promising by the authors. Grade 3 and higher irAEs were reported to be rare and self-limiting, with 14% which is promising when treatment feasibility is considered (81). In another phase 2 study, a patient group of nine who received pembrolizumab following high dose cytarabine was compared with a control group of 18 who underwent allogeneic HSCT and didn’t receive ICI. According to the one-year survival data, no significant difference was reported between the two groups (67% vs. 78%; p=0.34). For the group that received ICI, the 100-day mortality rate was 0%, while in the control group, it was 17%. Grade 3/4 acute GVHD risk didn’t increase in patients who received pembrolizumab prior to allogeneic HSCT while no indicator of chronic GVHD was reported (82). These findings support the aforementioned phase 2 study, in reflecting both the clinical activity and safety profile of cytarabine and pembrolizumab combination. Besides agents targeting PD-1/PD-L1 axis, anti-leukemic potential of humanized anti-TIM-3 antibody sabatolimab in combination with HMAs was investigated in 48 patients who were newly diagnosed with AML and ineligible for intensive chemotherapy (83). The overall response rate was reported as 40% while 30% of these patients achieved CR/CRi.

When ICIs’ role in maintenance is considered, preclinical studies indicate that ICIs can prevent leukemic cells’ evasion of the immune system and, thus, overcome tumor persistence. Another phase 2 study investigating efficacy of nivolumab on 14 high-risk AML patients in complete remission who were not eligible for allogeneic HSCT indicated that by the end of the year, 71% of patients were in complete remission, indicating the drug’s safety and feasibility in high-risk AML (84).

In brief, numerous recent clinical studies involving ICI as a single agent or combined with other treatments demonstrated promising results regarding clinical efficacy and safety profile. However, it is early to draw distinct conclusions about ICIs’ use in AML and further research is needed. It should be noted that currently, there is no ICI approved by the FDA in the treatment of AML, and the clinical trials regarding ICI in AML treatment are still at the early stages with results revealing modest efficacy, especially for monotherapy the refractory settings (85).

As mentioned earlier, chemotherapy in AML is divided into two phases; induction therapy and consolidation therapies which both vary according to the patient’s age, presence of co-morbidities and genetic alterations. Induction therapy aims to eliminate the blasts in the peripheral blood and to restore normal hematopoiesis while consolidation therapy is administered to remove residual leukemic cells (86). In clinical trials, efficacy of ICIs is mainly investigated in combination with chemotherapy agents and HMA (87). intervention in AML remains as allogeneic HSCT while the clinical trials involving ICI are ongoing and up-and-coming.

When considering treatments targeting BM microenvironment, CXCL12 (C-X-C motif chemokine ligand 12)/CXCR4 (C-X-C chemokine receptor 4) axis is the most studied pathway in AML treatment; as reported, inhibition of this pathway leads to mobilization of leukemic cells, sensitizes them to chemotherapy and promotes apoptosis (134–138). The anti-CXCR4 antibody ulocuplumab has shown to inhibit CXCL12-mediated cell migration and promote apoptosis in in vivo murine AML models as well as promoting chemosensitivity via mobilizing AML cells to circulation in clinical studies (139–141). Another common strategy is inhibiting the Wnt/β-catenin pathway to diminish the protection provided by BMM: β-catenin is highly expressed in unfavorable and relapsed AML patients, and Wnt/β-catenin inhibitor PRI-724 was shown to suppress cell growth while promoting apoptosis in AML blasts and stem/progenitors (142). Wnt/β-catenin/FLT3 inhibitor SKLB-677 promoted apoptosis in FLT3-driven AML both in-vitro, in-vivo and exvivo (143). Another Wnt/β-catenin inhibitor, BC2059 has shown promising results in treatment of AML stem or blast progenitor cells with FLT3 internal tandem duplication expression in combination with receptor tyrosine kinase inhibitors quizartinib and crenolanib (144).

Targeting adhesion molecules which support the leukemic cells is another approach in AML treatment. Being the receptor of vascular cell adhesion molecule (VCAM-1), integrin α4β1 (very late antigen 4 – VLA 4) plays role in the adhesion of leukemic myeloblasts to BMM (145). Humanized VLA-4 monoclonal antibody natalizumab has been reported to induce mobilization and sensitize leukemic cells to chemotherapy (146). In combination with cytarabine, VLA-4 inhibitor FNIII14 has shown to eradicate minimal residual disease in a murine AML model, underlining the importance of inhibiting cell adhesion-mediated drug resistance (147). By regulating VLA-4 avidity, adhesion molecule CD44 was shown to strengthen the connection between AML cells and BMM, thus, contributing to the supportive BMM (148). In a phase I study, humanized anti-CD44 monoclonal antibody RG7356 was found to be safe and well tolerated though it is not suitable as a monotherapy due to its’ limited clinical activity in AML treatment (149).

The endothelial cell adhesion molecule E-selectin is another important component of the vascular niche that regulates the balance between HSC renewal and commitment. However, the inflammatory mediators secreted by AML blasts increase the expression of endothelial niche E-selectin, which, in turn, promotes their survival and chemoresistance through AKT/NF-κB/mTOR signaling pathways (150). In an AML murine model bearing the human KMT2A-MLLT3 oncogene, the small molecule E-selectin mimetic GMI-1271/Uproleselan has enhanced the efficacy of AML treatment by overcoming vascular niche-mediated chemoresistance, indicating E-selectin blockade alleviates pro-survival signaling and improving therapeutic efficacy (150).

AML blasts modulate TME to enable disease progression, provide protection against therapeutic interventions and contribute to recurrence (151). In terms of ICPs, AML blasts can alter the T cell immunological synapses, promote inhibitory soluble factors to hamper T cell responses, and promote activity of MDSCs as well as promoting polarization of tumor associated macrophages (TAMs) to immunomodulatory M2 phenotype (151, 152). The interaction between AML cells and immune cells are visualized in Figure 2 .