Beyond Il-5: Metabolic Reprogramming and Stromal Support Are Prerequisite for Generation and Survival of Long-Lived Eosinophil

Eosinophils play surprisingly diverse roles in health and disease. Accordingly, we have now begun to appreciate the scope of the functional and phenotypic heterogeneity and plasticity of these cells. Along with tissue-recruited subsets during inflammation, there are tissue resident eosinophil phenotypes with potentially longer life spans and less dependency on IL-5 for survival. Current models to study murine eosinophils ex vivo rely on IL-5-sustained expansion of eosinophils from bone marrow hematopoietic progenitors. Although it does generate eosinophils (bmEos) in high purity, such systems are short-lived (14 days on average) and depend on IL-5. In this report, we present a novel method of differentiating large numbers of pure bone marrow-derived eosinophils with a long-lived phenotype (llEos) (40 days on average) that require IL-5 for initial differentiation, but not for subsequent survival. We identified two key factors in the development of llEos: metabolic adaptation and reprogramming induced by suppressed nutrient intake during active differentiation (from Day 7 of culture), and interaction with IL-5-primed stromal cells for the remainder of the protocol. This regimen results in a higher yield and viability of mature eosinophils. Phenotypically, llEos develop as Siglec-F(+)Ly6G(+) cells transitioning to Siglec-F(+) only, and exhibit typical eosinophil features with red eosin granular staining, as well as the ability to chemotax to eotaxin Ccl11 and process fibrinogen. This culture system requires less reagent input and allows us to study eosinophils long-term, which is a significant improvement over IL-5-driven differentiation protocols. Moreover, it provides important insights into factors governing eosinophil plasticity and the ability to assume long-lived IL-5-independent phenotypes.

Clinical-Grade Peptide-Based Inhibition of CK2 Blocks Viability and Proliferation of T-ALL Cells and Counteracts IL-7 Stimulation and Stromal Support

Despite remarkable advances in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), relapsed cases are still a major challenge. Moreover, even successful cases often face long-term treatment-associated toxicities. Targeted therapeutics may overcome these limitations. We have previously demonstrated that casein kinase 2 (CK2)-mediated phosphatase and tensin homologue (PTEN) posttranslational inactivation, and consequent phosphatidylinositol 3-kinase (PI3K)/Akt signaling hyperactivation, leads to increased T-ALL cell survival and proliferation. We also revealed the existence of a crosstalk between CK2 activity and the signaling mediated by interleukin 7 (IL-7), a critical leukemia-supportive cytokine. Here, we evaluated the impact of CIGB-300, a the clinical-grade peptide-based CK2 inhibitor CIGB-300 on T-ALL biology. We demonstrate that CIGB-300 decreases the viability and proliferation of T-ALL cell lines and diagnostic patient samples. Moreover, CIGB-300 overcomes IL-7-mediated T-ALL cell growth and viability, while preventing the positive effects of OP9-delta-like 1 (DL1) stromal support on leukemia cells. Signaling and pull-down experiments indicate that the CK2 substrate nucleophosmin 1 (B23/NPM1) and CK2 itself are the molecular targets for CIGB-300 in T-ALL cells. However, B23/NPM1 silencing only partially recapitulates the anti-leukemia effects of the peptide, suggesting that CIGB-300-mediated direct binding to CK2, and consequent CK2 inactivation, is the mechanism by which CIGB-300 downregulates PTEN S380 phosphorylation and inhibits PI3K/Akt signaling pathway. In the context of IL-7 stimulation, CIGB-300 blocks janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in T-ALL cells. Altogether, our results strengthen the case for anti-CK2 therapeutic intervention in T-ALL, demonstrating that CIGB-300 (given its ability to circumvent the effects of pro-leukemic microenvironmental cues) may be a valid tool for clinical intervention in this aggressive malignancy.

Direct Comparison of in-Vitro Growth of Primary Human Acute Myeloid Leukemia (AML) Cells in Stromal and Stromal-Free Conditions

Background: Acute myeloid leukemia (AML) is a very aggressive bone marrow malignancy which carries a poor prognosis despite intensive chemotherapy. The treatment of relapsed and refractory AML remains suboptimal. Although more novel therapies are being introduced for AML, there is a limitation of appropriate, predictive, preclinical models available to identify and test novel therapies. In-vitro drug sensitivity testing of patient-derived AML cells is increasingly being used to facilitate treatment options. However, these tests are often done in suboptimal conditions with difficulty in the interpretation of results. Accumulating evidence has shown that the long-term, in-vitro, survival of primary AML cells can be supported with stromal co-culture, which would also take into account the influence of the surrounding tumor microenvironment. However, there has been no direct comparison of a stromal co-culture method to non-stromal growth method of primary AML cells. This is the first comprehensive direct comparison of the proliferation and immunophenotypes of primary AML cells under two different stromal conditions and cytokines, the results of which would not only highlight the importance of stromal cells to chemotherapy resistance but also lay the groundwork for the feasibility and importance of comparing it to non-stromal drug sensitivity testing. Methods: The proliferation and immunophenotypes of seven bio-banked, relapsed/refractory primary AML peripheral blood samples were assessed in 6-well plates under four culture conditions: 1) cell culture media only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned media or 4) in a cocktail of four cytokines. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 were determined by flow cytometry weekly for 2 weeks. Results: Our results demonstrated a heterogeneous growth response among the seven patient samples in response to the four growth conditions. However, the end effect was that cytokines, HS-5 conditioned media and HS-5 stromal cells can all support the long-term proliferation and viability of the majority of primary AML samples. These three conditions showed statistically significant higher growth compared to the same primary cells cultured in media alone (p-values < 0.05). The degree of expansion was still significantly higher for those cells with cytokines than those with HS-5 conditioned media or stromal cells. Six of the 7 patient samples expanded with cytokine, up to 4-fold by 2 weeks of culture. In comparison 5 of the 7 patient samples expanded up to 3-fold with HS-5 stromal cells, and 4 of the 7 patient samples also expanded up to 3-fold with HS-5 conditioned media at 2 weeks of culture. For the 4 patients, whose cells were supported by both stromal cells and conditioned media, the degree of expansion was similar. Unexpectedly, one out of 7 patient samples had significantly higher expansion compared to all of the other patients in all growth condition, with a 9-fold expansion in cytokine and conditioned media, and a 12-fold expansion in HS-5 stromal cells. There was also a 3-fold expansion in media alone. Baseline AML immunophenotypes were preserved after 2 weeks of culture for all 4 growth conditions, with minimal differentiation. Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that each experimental condition alone can expand primary AML cells. In addition, cytokine support alone can expand primary AML cells at 2 weeks of culture for the majority of patient samples, independent of stromal support, a finding that has previously not been reported. Consistent with previous studies, our data also supports the conclusion that stromal support maintains long-term in-vitro expansion of primary AML cells. All three experimental conditions showed an overall effect of growth with high viability and maintenance of immunophenotypes at 2 weeks of culture. The direct comparison of drug screening under these three experimental conditions would permit the exploration of the protective effect of the bone marrow microenvironment on AML cells allowing for the identification of individualized therapeutic options, while also allowing for comparison to non-stromal based in-vitro assays. Disclosures No relevant conflicts of interest to declare.

The hallmarks of cancer

A major challenge for cancer medicine involves the remarkable variability of the disease, at all levels. The hallmarks of cancer constitute an organizing principle that may provide a rational basis for distilling this complexity so as to better understand mechanisms of the disease in its diverse manifestations. The conceptualization involves eight acquired capabilities—the hallmarks of cancer—and two generic characteristics of neoplastic disease that facilitate their acquisition during the multistage process of neoplastic development and malignant progression. The integration of these hallmark capabilities in symptomatic disease involves multiple cell types populating the tumor microenvironment, including heterogeneous populations of cancer cells, in particular cancer stem cells, and three prominent classes of stromal support cells. A premise is that the hallmarks of cancer constitute a useful heuristic tool for understating the mechanistic basis and interrelationships between different forms of human cancer, with potential applications to cancer therapy.

Functional Inhibition of Mesenchymal Stem and Progenitor Cells (MSPC) Significantly Contributes to Hematopoietic Insufficiency with Acute Myeloid Leukemia (AML)

Pancytopenia is the most prominent clinical finding in patients with acute myeloid leukemia (AML) and represents a major cause for morbidity and mortality. So far, the underlying mechanisms leading to hematopoietic insufficiency in AML are poorly understood and therefore often mechanistically summarized as marrow replacement by infiltrating leukemic cells. Mesenchymal stem and progenitor cells (MSPC) are integral components of the bone marrow (BM) microenvironment and play an indispensable role for the regulation of normal hematopoiesis. Two AML mouse models have recently shown, that expansion of the leukemic clone leads to numeric changes and functional disturbances of niche components such as MSPC and osteoblasts, resulting in insufficient hematopoietic support (Hanoun et al. 2014; Frisch et al. 2012). As the knowledge about MSPC in human AML is limited so far, we conducted a detailed analysis of AML-derived MSPC in order to elucidate their contribution to hematopoietic failure. For this purpose we investigated the molecular and functional properties of BM-derived MSPC of 46 patients with AML covering all relevant subtypes according to WHO classification at diagnosis and/or during course of disease and compared them with MSPC functions of healthy controls. Hematopoietic insufficiency in the 31 patients at diagnosis was mirrored by 76% of the patients having bi- or pancytopenia and by median ANC of 1183/μl, median hemoglobin of 9.0 g/dl, and median platelet count of 67.000/μl. MSPC of these newly diagnosed patients exhibited significantly impaired growth capacities as shown by an altered morphology, reduced CFU-F activity, a lower number of passages and cumulative population doublings. While adipogenic differentiation potential was not affected, osteogenic differentiation potential of AML-derived MSPC was significantly reduced as indicated by cytochemical stainings, reduced Osterix and Osteocalcin (OC) mRNA levels as well as OC serum levels. Furthermore we detected altered mRNA and/or protein expression of key molecules involved in the regulation of hematopoietic stem and progenitor cells (HSPC), namely SCF, Angiopoietin-1, Jagged1 and Osteopontin. Functionally, this translated into a significantly diminished ability of AML-derived MSPC to support healthy CD34+ HSPC in LTC-IC assays. This insufficient stromal support was reversible and correlated with disease status, as LTC-IC frequency returned to normal values in patients in remission, but remained low in patients with refractory disease. Along with this we also observed a significant increase of OC serum levels in patients, who achieved complete remission. These data clearly suggests a direct causal relationship between the presence of leukemic cells and MSPC functionality. In further support of this idea we observed reduced proliferation and osteogenic differentiation of healthy MSPC following cultivation in conditioned media (CM) of 4 AML cell lines (THP-1, HL-60, MV4-11, MOLM-13). A comparable inhibitory effect of AML cells on healthy MSPC growth was seen in transwell-assays arguing in favor for a cell-contact independent mechanism. In summary, our data show that AML-derived MSPC are structurally and functionally altered resulting in an insufficient stromal support for normal hematopoiesis in AML. The correlation between clinical remission status and stromal support function together with the finding, that healthy MSC can adopt an AML-like phenotype when exposed to AML-CM suggest an instructive role of the leukemic precursor cells. Disclosures Dührsen: Celgene: Honoraria, Research Funding. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.