While many patients diagnosed with acute myeloid leukemia (AML) respond to initial treatment, relapse remains the major obstacle to cure. The application of chemotherapy to relapsed AML is unlikely to yield long-term responses, so alternative modalities are sorely needed. Hematopoietic stem cell transplant (HSCT), the only curative therapy for relapsed AML, relies on the combination of high-dose chemotherapy and the graft-versus-leukemia (GVL) effect. T-cell activation is a critical feature of GVL and appears to restore long-lasting tumor immune surveillance in a variety of otherwise fatal hematologic malignancies. Unfortunately, the often short-term clinical activity observed in trials of T-cell-activating therapies suggests that T-cell activation alone is insufficient to recreate the lasting antitumor immunity that characterizes successful GVL.
A growing number of studies suggest the inflammatory milieu created by AML drives AML progression and directly limits T-cell antitumor reactivity. This new understanding is driving the development of a new generation of immunotherapies that incorporates “inflamodulation” to combat or reverse the immunomodulatory effects of the AML inflammatory environment.
The Critical Role of T Cells in GVL
The foundational role T cells play in support of the clinical activity of HSCT is clear.1 The additional infusion of T cells in the context of donor lymphocyte infusion can rescue failed HSCTs, while depleting grafts of T cells increases the risk of relapse.2 These data are buttressed by the ability to isolate donor-derived, leukemia-reactive, activated cytotoxic T lymphocytes from patients after HSCT capable of eliminating leukemic stem cells.3,4 Together, these experiments suggested that if only T cells could be sufficiently activated, anti-AML tumor surveillance could be restored and lead to durable cures without the side effects of transplant. Efforts to engineer leukemia-specific T-cell activation were attempts to make good on this promise.
The immune environment of AML opposes these efforts. At diagnosis and relapse, endogenous T cells within patients with AML are reactive to tumor antigens but exert ineffective immune responses.5 In some ways, this is tautology: if immune surveillance were effective, AML would not manifest in the first place. Nonetheless, identification of leukemia-reactive T-cell receptor (TCR) clones6-8 and high-dimensional mapping of T-cell states9,10 from leukemic and post-treatment bone marrow samples has allowed the identification of mechanisms that stifle effective immune responses. More broadly, transcriptional profiling of large patient cohorts suggests that approximately 25% of patients with AML exhibit immune states that modulate patient outcomes.11-14 These include proliferation of regulatory T cells15 and accumulation of exhausted16-18 or anergic19 cytotoxic T lymphocytes, some of which may be reversible,5 suggesting an avenue to therapeutic intervention. Efforts to modulate this immune inflammatory environment for therapeutic effect are called “inflamodulation.”
Three approaches to synthetic leukemia-specific T-cell activation in patients are now maturing: (1) multispecific T-cell-activating antibodies, (2) transgenic delivery of engineered TCRs and chimeric antigen receptors (CARs), and (3) immune checkpoint blockade. The first two of these technologies share the strategy of providing synthetic antigenic signaling, while the third removes negative signals that suppress responses already present.
The clinical experience of flotetuzumab (MGD006) in a phase I/II trial supports the claim that T-cell activation alone is insufficient to induce lasting remissions in most patients.20 While expression of CD123 was required for trial entry, the overall rate of complete remission (CR) or CR with incomplete hematologic recovery was only 18% following treatment with an optimized drug dosing scheme. However, remission was more likely in patients with bone marrow transcriptional gene signatures associated with neutrophil and macrophage infiltration and interferon (IFN)-γ production, even in patients with the usually dire TP53 mutations.21 These data support that combining T-cell activation with the proper inflammatory milieu is a requirement for producing the long-term remissions observed following transplant with hoped-for frequencies.
Evaluation of CARs targeting CD33,22,23 CD123,24,25 CLL1,26 CD7,27 FLT3,28 and other antigens seems to further undermine claims that the root cause of failure is the lack of a suitable target antigen. Constructs targeting each of these antigens produce T-cell activation in vitro and in preclinical models but have not yet demonstrated clinical activity. As a result, it seems that only B-cell malignancies, including lymphocytic leukemia, lymphomas, and myeloma exhibit the proper environment to sustain long-term tumor immune surveillance following synthetic T-cell activation.
New Approaches to Immune-Environmental Engineering
“Immunotherapists” have recognized that T-cell activation alone is insufficient to overcome the powerful immune-suppressive effects of AML. As a result, novel classes of immune-active therapies that attempt to either reverse the effects of the inflammatory AML microenvironment or supply missing signals that sustain T-cell survival and antitumor activity are being evaluated.
Inflamodulation is increasingly recognized as a successful strategy for treating myeloproliferative neoplasms. The high frequency of JAK2 mutations and the clinical activity of JAK 2 inhibitors have led some investigators to explore combinations of JAK2 inhibition with AML-type therapies. Small studies have found the doublet combination of ruxolitinib with hypomethylating agents is seemingly rarely effective for the treatment of relapsed or refractory patients.29,30 Nonetheless, while JAK2 mutations are infrequent among patients with AML, activation of down stream JAK2 targets, such as STAT3, is common.31 Thus, more specific and potent T-cell-activating strategies will be needed.
Super-charged T cells are coming, and the modularity of CAR designs leads these adoptive cell therapies to the forefront of inflamodulatory testing. Incorporating a secreted anti-IL6 antibody into the CAR design not only eliminated neurotoxicity and made cytokine release syndrome a rare or limited event, but also maintained high response rates (90% among the acute lymphocytic leukemia cohort).32 Incorporation of secreted IL15 may provide both pro-survival and anti-immunosuppressive signals, thereby reversing the effects of myeloid-derived suppressor cells,33 which are increased in AML.34 Secreted IL15,35 membrane-bound IL15,36 and injected IL15-polymer conjugates37 are therefore being explored.
While cytokines create a pro-survival milieu, a super-charged receptor “engine” may break through environmental limitations of T-cell responses. Traditional CAR designs contain CD3 and 4-1BB signaling domains, and “strapping on” additional signaling domains like CD40 may boost antigenic signals, while activation of MYD88 may favorably tune metabolism.38 The number of these studies and their rapid pace suggest CAR technology is an ideal model and platform for evaluating the signals needed to generate lasting GVL.
Attempts to simultaneously limit inflammation and enhance T-cell reactivity may be generalized through modulation of IFNγ. In the case of flotetuzumab, IFNγ production is a strong marker of T-cell activity and balances antihost with antileukemic allo-reactivity;39,40 therefore, modulating IFNγ is a promising strategy to boost GVL and antileukemic responses.41 Boosting IL18 signaling is one novel and promising strategy. A potent soluble inducer of IFNγ, IL18 activity can be supplemented by providing a synthetic decoy-resistant IL18 (DR18),42 while simultaneously removing exhausted T cells via administration of cyclophosphamide following haploidentical transplants43 or providing memory cells through infusion of donor lymphocytes. It is uncertain whether this strategy seeking to supercharge GVL can be translated to clinical testing without undue toxicity.
The Best Weed Suppression Is a Healthy Crop
The inflammatory environment produced by AML not only limits T-cell antitumor surveillance but also drives disease progression. Working in zebrafish models of clonal hematopoiesis of indeterminant potential (CHIP), the laboratory of Lenard Zon44 showed that inflammatory signals elaborated by CHIP clones limited the growth of normal hematopoietic stem cells (HSCs). Genetic evaluation of preleukemic CHIP clones revealed evolved resistance to inflammatory signals. These findings suggested a positive feedback loop in which preleukemic clones and leukemic stem cells are simultaneously a source of and resistant to inflammatory signals, setting up an environment that maintains an advantage for AML cells over residual normal HSCs.
Expression of genes responsive to inflammatory signaling is predictive of chemotherapy resistance45 and improves risk stratification.46 Nearly half of AMLs are enriched in expression of genes associated with inflammation.47 In one study, mechanistic interrogations using short hairpin RNA (shRNA) screens found that mediators of inflammatory signaling were standout targets,47 with hits converging on MYB and SPI/PU.1, which function as “master switches” linking monocytic differentiation with inflammatory signaling.48 Additional shRNA screens also targeted an unstudied gene, IRF2BP2, which, upon investigation, similarly functions to repress inflammatory signaling. Findings that IRF2BP2 knockdown restored sensitivity to TNFα and IL1β and promoted AML apoptosis confirm the generalized model and have since been supported by a growing number of studies, wherein resistance to inflammatory signals is a critical component of the AML microenvironment.
Attempts to synthetically engineer the positive lasting immune activation seen during GVL following HSCT without graft-versus-host disease is a worthy, if incompletely realized, goal. Is therapeutic modulation of the AML inflammatory microenvironment the missing key to engineering GVL-like AML responses? Such “inflamodulation” is an underexplored area of clinical research and tempting to pursue. The next wave of immunotherapies will be the first to explore applications of this new understanding.
Jerald Radich, MD, is a Professor in the Clinical Research Division and Kurt Enslein Endowed Chair at Fred Hutch.
Jacob Appelbaum, MD, PhD, is an Acting Instructor in the Division of Hematology at the University of Washington School of Medicine.
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