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-X
inhibitors to activate apoptosis. We demonstrate the translational potential of an AMPK and BCL-2/BCL-X
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.
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.
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
model.
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
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.
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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The original version of this Article contained an error in Fig. 7. In panel b, the survival curves were shifted relative to the y axis. This error has been corrected in both the PDF and HTML versions ...of the Article.
Abstract
Elevated MYC levels sensitize 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 dramatically synergizes with BCL-2/BCL-XL inhibitors to activate MYC-dependent 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 either navitoclax or venetoclax efficiently inhibits tumor growth, confers survival benefits and induces tumor infiltration by immune cells. However, withdrawal of the drugs allowed tumor re- growth with presentation of PD1+/CD8+ T cell infiltrates, suggesting immune escape. A two-step treatment regimen, beginning with neoadjuvant metformin+venetoclax to induce apoptosis and followed by tumor resection and adjuvant metformin+venetoclax+anti-PD1 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.
Citation Format: Heidi M. Haikala, Johanna M. Anttila, Mariel Savelius, Elsa Marques, Tiina Raatikainen, Mette Ilander, Henna Hakanen, Johanna Mattson, Paivi Heikkila, Marjut Leidenius, Heikki Joensuu, Satu Mustjoki, Panu Kovanen, Martin Eilers, Joel D. Leverson, Juha T. Klefström. Pharmacological reactivation of MYC-dependent apoptosis cooperates with anti-PD1 immunotherapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-189.
Abstract
Anti-PD1 therapy has proven to be effective in various cancer types, but not all patients benefit from the therapy. Further, no comprehensive immunologic monitoring during anti-PD1 treatment ...has yet been published. In this study, we aimed to discover the effects of anti-PD1 therapy on the immune system, especially on NK and NKT-cells, which are less studied, but known to be involved in antitumor immune events. Peripheral blood samples from immuno-oncology (IO) naive metastatic melanoma patients (n=20) were obtained before the first infusion of pembrolizumab or nivolumab (D0), then 1 and 3 months after the initiation of treatment. From each time-point, complete blood counts (CBC) were obtained, and comprehensive immunophenotyping of NK, NKT, and T-cells was performed with multicolor flow cytometry. Moreover, 92 different serum cytokines were measured using the Olink inflammation panel. The protein levels are presented as arbitrary units of normalized protein expression, NPX, on Log2 scale. The CBC revealed that the proportion of lymphocytes (mean D0 30.6% vs. 3mo 24.9%, p=0.02), decreased during the treatment but no changes were observed in absolute numbers or in other leukocytes. Immunophenotyping of lymphocyte subpopulations revealed that the frequency of NKT brighT-cells was increased (D0 1.8% vs. 1mo 2.3%, p=0.01) and that cytotoxic NK CD56dim cells expressed more CD25 (D0 19.5% vs. 1mo 23.7%, p=0.02) and CD45RO (D0 14.7% vs. 1mo 21.1%, p=0.03) surface markers after 1 month of therapy. The cytokine assay indicated that during anti-PD1 treatment the levels of CXC family cytokines were increased in the serum; CXCL9 (D0 470 vs. 1mo 1070, p=0.0003, D0 470 vs. 3mo 1227, p=0.0007), CXCL11 (D0 49.8 vs. 1mo 81.8, p=0.005), CXCL10 (D0 1000 vs. 1mo 2078, p=0.0003). Also, an increase in IL-12B (D0 31.6 vs. 1mo 41.2, p=0.003, D0 31.6 vs. 3mo 32.4, p=0.04) and TNFRSF9 (D0 126.4 vs. 1mo 181.0, p=0.01, D0 126.4 vs. 3mo 149.0, p=0.04) levels was observed.To further examine these results, patients were categorized into two cohorts: responders (R, n=6, PFS=17.0 months) and patients with progressive disease (PD, n=9, PFS=5.0 months) in terms of the duration of progression-free survival (PFS) and decrease in tumor burden. 5 patients were excluded due to challenging clinical evaluation of response. When examining the differences between these cohorts, CBC indicated a significant decrease in the mean frequency of lymphocytes in PD (D0 26.9% vs. 3mo 19.2%, p=0.04), but not in the R cohort. The responders had also higher frequency of lymphocytes at 1- and 3-month time-points (R 34.5% vs. PD 25.4%, p=0.02, R 32.4% vs. PD 19.2%, p=0.01, respectively) and lower frequency of neutrophils before initiation and after 1 and 3 months of treatment (R 50.8% vs. PD 58.6%, p=0.04, R 45.3% vs. PD 59.4%, p=0.01, R 49.8% vs. PD 64.5%, p=0.04, respectively). The CBC absolute counts revealed that the responders had less neutrophils (R 2.8 109/L vs. PD 4.9 109/L, p=0.04) and monocytes (R 0.4 109/L vs. PD 0.7 109/L, p=0.04) after 3 months of treatment when compared to PD. The immunophenotyping showed that responders had more NKT dim cells before (R 10.1% vs. PD 3.5%, p=0.03) and after 3 months of therapy (R 15.7% vs. PD 3.7%, p=0.03) and the increase in NKT bright frequency was observed only in R (D0 2.5% vs. 1mo 3.4%, p=0.04) and not in PD cohort. The cytokine assay indicated that the CXCL9 was increased in R cohort (D0 476.8 vs. 1mo 1480.2, p=0.01), but not in PD, making the cytokine levels greater in R (R 1480.2 vs. PD 639.3, p=0.01) after 1 month of therapy. Based on our preliminary results, we believe that high frequency of NKT-cells in blood is related to positive treatment response, and in addition to T-cells, also NK and NKT-cells may play a key role in the antitumor response induced by PD-1 inhibition. Hence, further research on the effect of anti-PD1 therapy on NK and NKT-cells is needed to better understand their role in positive therapy response.
Citation Format: Henna H.E. Hakanen, Micaela Hernberg, Siru Mäkelä, Bhagwan Yadav, Oscar Brück, Susanna Juteau, Laura Kohtamäki, Mette Ilander, Satu Mustjoki, Kreutzman Anna. Metastatic melanoma patients responding to PD1 therapy have higher proportion of peripheral blood NKT-cells abstract. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A130.
Abstract
Despite the impressive impact of CTLA4 and PD1-targeted immuno-oncologic (IO) therapies, a large proportion of patients fail to respond. The observed variance in treatment efficacy has been ...linked to heterogeneity in the immune cell distribution of individual patients. Lymphocyte activation gene 3 (LAG3, CD223) is one of the newest IO targets entering the clinic. Triggering of LAG3 on T-cells by HLA-DR -ligands has a well-established role in the negative regulation of T-cell function. Although for example on activated regulatory T-cells (Treg) LAG3 is widely expressed, the detailed effects of LAG3 on various cell types are still unknown. We profiled over 30,000 CD45+ lymphocytes cells using a novel paired single-cell RNA and T-cell receptor (TCR) αβ chain (10x Genomics) sequencing method for peripheral blood samples from two patients receiving anti-LAG3 and anti-PD1 combination treatment in multicentre phase I trial. The sequenced cells were from IO-treatment naïve metastatic melanoma patients from before, after 4 weeks and after 12 weeks of the start of therapy. For validation, we performed TCRβ-sequencing and flow cytometry analysis of a larger cohort of melanoma patients (n = 12) enrolled in the same study. To gain in-depth understanding of the immune cell subsets, we used a recently described method to seek matching mutual neighbors between patients to normalize the interpatient variation to enable a systematic comparison across patients. To identify phenotypic clusters, we used a graph theory-based clustering method SNN-Cliq and built predictive machine learning classifiers to assess the reproducibility of learnt clusters. After optimizing our choice of input genes and parameters, we identified 16 distinct clusters that define the T-cell roadmap of anti-LAG3 and anti-PD1 treatment that includes 6 CD8+, 8 CD4+ (including Treg cluster), and 2 other clusters. We identified 4 CD8+ T-cell clusters of increasing cytotoxicity profile using a cytotoxicity score and the pseudotime algorithm Monocle3. The most highly cytotoxic clusters increased and a cluster of lower cytotoxicity score decreased during the treatment. In addition, we defined 2 CD8+ exhaustion clusters. During treatment, we observed a decrease in the exhausted T-cell cluster defined by LAG3 and PDCD1 expression, but an increase in TIGIT+ exhaustion cluster. Furthermore, ordering the cluster of FOXP3+ Treg cells along pseudotime trajectory revealed two different fates for Treg cells, where the other fate showed significantly decreased expression of LAG3 and PDCD1, adding evidence of the effect of the treatment on Treg cells. TCRαβ analysis revealed 19 individual expanded clonotypes of size of 100 sequenced individual cells. The true transcriptomic heterogeneity of identical clonotypes was revealed as most clonotypes spanned several of the 16 different clusters, challenging our view of clonal expansion. Furthermore, we were able to assess the temporal phenotypic changes in individual clones during the course of immunotherapy. Most clonotypes, including cytotoxic and exhausted clones, had more homogenous transcriptomes before the start of the treatment, but diversified during the therapy, suggesting a release of immunologic break in these clones. In summary, we defined the evolution of immunological response to anti-LAG3 and anti-PD1 therapy cell by cell, and described an increase in the heterogeneity of cytotoxicity in CD8+ cells, distinct fates of Treg cells and the release of transcriptomic profiles of individual T-cell clonotypes during IO treatment.
Citation Format: Jani Huuhtanen, Henna H.E. Hakanen, Tapio Lönnberg, Olli Dufva, Katriina Peltola, Siru Mäkelä, Micaela Hernberg, Petri Bono, Kreutzman Anna, Satu Mustjoki. Single-cell roadmap of the evolution of T-cell response during anti-LAG3 and anti-PD1 combination treatment in metastatic melanoma patients abstract. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A134.
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