Denise Wolleschak

Clinically relevant doses of FLT3-kinase inhibitors quizartinib and midostaurin do not impair T-cell reactivity and function.

The vast majority of acute myeloid leukemia (AML) patients harboring an FLT3-ITD mutation experience relapse within a short period of time after discontinuation of chemotherapy.[1][1] Treatment options include experimental trials using FLT3-tyrosine kinase inhibitors (TKI) or allogeneic stem cell

Specificity of JAK-kinase inhibition determines impact on human and murine T-cell function

DOX (Supplementary Figure 7C). For in vivo therapy studies, mice bearing orthotopic MM tumors were treated with systemic administration of ApDCs or equal molar free DOX (Figure 2a). Changes in MM tumor size were monitored by whole-body bioluminescence imaging scans every 6 days (Figure 2b). Images at day 36 showed that ApDC treatment markedly inhibited orthotopic tumor growth, whereas free DOX had no effect under the same treatment conditions comparing to control group (Figure 2c). In addition, Kaplan–Meier survival analysis revealed that ApDCs treatment significantly improved survival rates of mice bearing MM tumors, indicating that ApDC was able to specifically deliver DOX to MM cells and accumulate high-DOX concentrations selectively within MM tumors for targeted therapy, although a sub-toxic concentration of DOX was administered to avoid off-target side effects. For histological examination, fresh cells from tumors were collected and cellular green fluorescent protein expression was detected by fluorescent microscopy (Figure 2e). Immunostaining of tumor tissues confirmed that MM tumor cells were CD38-positive and restricted for immunoglobulin lambda light chain expression (Figure 2f). Finally, analysis of healthy immunocompetent mice showed that ApDC treatment did not induce inflammation (Figure 2g), indicating suitability for in vivo therapy. Taken together, our study demonstrates that ApDCs are a new targeted therapeutic approach for specific delivery and intracellular release of a high drug payload under a unique pH-controlled mechanism in MM cells (Figure 1a). Our findings also indicate the clinical potential of ApDCs, which not only selectively inhibit MM tumors, but also have little or no off-target side effect in patients. To initiate clinical trials, further improvements and additional preclinical testing may be needed as discussed previously. Interestingly, this ApDC technology can be used as a universal platform to treat many cancers by simply replacing the aptamer sequence with one that targets different tumor biomarkers.

Chronic myelogenous leukemia evolving after treatment of multiple myeloma

![Figure][1] A 51-year-old woman presented with bone pain, severe anemia, and thrombocytopenia (hemoglobin 4.02 g/dL; platelets [PLT] 121 × 109/L). Laboratory evaluation revealed monoclonal M-spike and elevated immunoglobulin G of 43.8 g/L. Giemsa-staining of the bone marrow (BM) aspirate

The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

JAK2-V617F activates β1-integrin-mediated adhesion of granulocytes to vascular cell adhesion molecule 1

JAK2-V617F activates β1-integrin-mediated adhesion of granulocytes to vascular cell adhesion molecule 1

Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML

Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death. Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort. Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy. Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final “common path.” Clin Cancer Res; 23(16); 4805–16. ©2017 AACR.

Expression and function of ABC-transporter protein ABCB1 correlates with inhibitory capacity of Ruxolitinib in vitro and in vivo

The development of Janus kinase (JAK) inhibitors has revolutionized the therapeutic landscape for the treatment of myeloproliferative neoplasms (MPN). Therefore, improving the efficacy and safety of JAK inhibitor therapy is a major prerequisite for reliable clinical use in the future. Ruxolitinib (

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation

JAK2-V617F–positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, &bgr;1 and &bgr;2, may contribute to CMN pathophysiology remained unclear. &bgr;1 (&agr;4&bgr;1; VLA-4) and &bgr;2 (&agr;L&bgr;2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1– (VCAM1-) and intercellular adhesion molecule 1–coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of &bgr;1 and &bgr;2 integrins for their respective ligands. For &bgr;1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti–VLA-4 and anti–&bgr;2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-&bgr;2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.

Cell autonomous expression of CXCL-10 in JAK2V617F-mutated MPN

PurposeMyeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem- and progenitor cells. Mutation of Janus-Kinase 2 (JAK2) is the most frequent genetic event detected in Philadelphia-negative MPN. In advanced phases, the clinical hallmark of the disease is a striking inflammatory syndrome. So far, the cellular and molecular basis of inflammation is not fully understood. We, therefore, sought to investigate the relationship of activating JAK2 mutation and aberrant cytokine expression in MPN.MethodsCytokine array was performed to identify Jak2V617F-related cytokine expression and secretion. CXCL10 mRNA expression was analyzed by qPCR in peripheral blood cells. To exclude paracrine/autocrine stimulation as a potential mechanism, we generated Ba/F3-EpoR-JAK2WT or EpoR-JAK2V617F cells lacking CXCL10 receptor. Pharmacologic inhibition of JAK2 kinase was achieved by JAK-Inhibitor treatment. Signaling pathways and downstream effectors were characterized by Western blotting, immunofluorescence microscopy, luciferase reporter assays, qPCR, and chromatin-immunoprecipitation studies.ResultsWe identified CXCL10 as the most highly induced cytokine in JAK2-mutated cell lines. In MPN patients, CXCL10 is highly expressed in JAK2V617F but not JAK2WT MPN or healthy donor controls. Moreover, CXCL10 expression correlates with JAK2V617F allelic burden. High CXCL10 correlates with the presence of clinical risk factors but not with clinical symptoms and quality of life. Pharmacologic inhibition of mutated JAK2 kinase inhibits CXCL10 expression. NFκB signaling is activated downstream of JAK2V617F receptor and directly induces CXCL10 expression.ConclusionsOur data provide first evidence for a link between oncogenic JAK2V617F signaling and cell intrinsic induction of CXCL10 induced by activated NFkB signaling.