Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

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Elevated MYC expression sensitizes tumor cells to apoptosis but the therapeutic potential of this mechanism remains unclear. We find, in a model of MYC-driven breast cancer, that pharmacological activation of AMPK strongly synergizes with BCL-2/BCL-XL inhibitors to activate apoptosis. We demonstrate the translational potential of an AMPK and BCL-2/BCL-XL co-targeting strategy in ex vivo and in vivo models of MYC-high breast cancer. Metformin combined with navitoclax or venetoclax efficiently inhibited tumor growth, conferred survival benefits and induced tumor infiltration by immune cells. However, withdrawal of the drugs allowed tumor re-growth with presentation of PD-1+/CD8+ T cell infiltrates, suggesting immune escape. A two-step treatment regimen, beginning with neoadjuvant metformin+venetoclax to induce apoptosis and followed by adjuvant metformin+venetoclax+anti-PD-1 treatment to overcome immune escape, led to durable antitumor responses even after drug withdrawal. We demonstrate that pharmacological reactivation of MYC-dependent apoptosis is a powerful antitumor strategy involving both tumor cell depletion and immunosurveillance.

The immunologic microenvironment in various solid tumors is aberrant and correlates with clinical survival. Here, we present a comprehensive analysis of the immune environment of acute myeloid leukemia (AML) bone marrow (BM) at diagnosis. We compared the immunologic landscape of formalin-fixed paraffin-embedded BM trephine samples from AML (n = 69), chronic myeloid leukemia (CML; n = 56), and B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 52) at diagnosis to controls (n = 12) with 30 immunophenotype markers using multiplex immunohistochemistry and computerized image analysis. We identified distinct immunologic profiles specific for leukemia subtypes and controls enabling accurate classification of AML (area under the curve [AUC] = 1.0), CML (AUC = 0.99), B-ALL (AUC = 0.96), and control subjects (AUC = 1.0). Interestingly, 2 major immunologic AML clusters differing in age, T-cell receptor clonality, and survival were discovered. A low proportion of regulatory T cells and pSTAT1+cMAF− monocytes were identified as novel biomarkers of superior event-free survival in intensively treated AML patients. Moreover, we demonstrated that AML BM and peripheral blood samples are dissimilar in terms of immune cell phenotypes. To conclude, our study shows that the immunologic landscape considerably varies by leukemia subtype suggesting disease-specific immunoregulation. Furthermore, the association of the AML immune microenvironment with clinical parameters suggests a rationale for including immunologic parameters to improve disease classification or even patient risk stratification.

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8 þ T cells in PB, together with a higher amount of tumor-infiltrating CD8 þ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4 þ and CD8 þ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials.

Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.Experimental Design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model.Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell-cell contacts, altered cell-matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions.

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Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in TMEM173 and IFIH1 showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways in vitro, and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in TMEM173 and IFIH1 as likely modifiers of the phenotype.