ALBERT

Description: Key Points We provide a functional analysis of IGF1R expression in primary human B-CLL. Sorafenib reduces IGF1R expression in B-CLL.

Description: The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

Description: Previous work has shown that FLT3-ITD mediated leukemogenesis is associated with increased expression of PIM1 and PIM2 serine/threonine kinases. Here we show that retroviral expression of FLT3-ITD could not compensate impaired clonogenic in vitro growth of PIM1−/− bone marrow cells. Induction of a lethal myelo- and lymphoproliferative disorder by FLT3-ITD in vivo was independent of PIM2, but rather unexpectedly, lethally irradiated recipients could not be reconstituted with FLT3-ITD expressing bone marrow cells lacking PIM1. Transplants of CSFE-labeled PIM1−/− cells revealed an impaired homing capacity to bone marrow and spleen. Expression of lower surface CXCR4 levels (while maintaining normal total CXCR4 levels) in PIM1−/− bone marrow cells was associated with significantly reduced migration towards a CXCL12 gradient and impaired CXCL12-mediated intracellular Ca2+ release. Using siRNA-mediated knockdown, a small molecule PIM inhibitor, expression of a dominant-negative acting PIM1 mutant or re-expression of PIM1 in knockout cells, we observed that PIM1 activity was critical for CXCR4 surface expression. In vitro kinase assays and masspectrometric analysis further revealed that PIM1 directly phosphorylated serine 339 located in the CXCR4 intracellular domain known to be essential for proper receptor recycling. Interestingly, in leukemic blasts from acute myeloid leukemia (AML) patients, we found an association of increased PIM1 expression and high-level of surface CXCR4. In addition, treatment of the cells with a small molecule PIM inhibitor resulted in decreased surface CXCR4 expression in some patients. Our work suggests that PIM1 exerts its oncogenic activity not only by supporting proliferation and survival but also by regulation of cell homing and migration through direct modification of the CXCL12/CXCR4 axis. As CXCR4 is a key mediator of cancer stem cell homing and metastasis, targeting of PIM1 may offer new therapeutic avenues against tumor progression and relapse.

Description: The hedgehog pathway (Hh) is one of several key developmental pathways whose deregulation is known to induce tumor formations in mice and humans including medulloblastoma or basal cell carcinoma. Certain human solid tumors (prostate cancer, pancreatic cancer) have been identified which require sustained Hh-Gli signaling for proliferation. Here, we show for the first time a crucial role of Hedgehog signalling in hematopoietic malignancies. In our experiments, we demonstrate that the Hh pathway is essential for survival of more than 70% of primary lymphomas extracted from transgenic Eμ-Myc mice or Cdkn2a−/− mice (INK4Arf−/−). 34 Myc-lymphomas (10 B-cell lymphomas, 12 plasmablastomas, 10 plasmocytomas, 2 mixed lymphomas) and 10 Cdkn2a−/− lymphomas were isolated from bone marrow, spleen or lymph nodes from diseased mice and expanded on bone marrow stroma cells from Cdkn2a−/− mice. Inhibition of Hh signaling by Cyclopamine (2–5 μM), an alkaloid which inhibits smoothened activation, induced apoptosis in 80% of B-cell lymphomas, 82% of plasmablastomas and 60% of multiple myelomas without affecting stroma proliferation. Protein or transcript levels of Hh pathway downstream targets, such as Gli1, Ptch or Bcl2 but not BMI1 were downregulated after 24h treatment with Cyclopamine. Overexpression of Bcl2 or Gli3 could inhibit apoptosis induction by Cyclopamine. High levels of Ihh protein could be detected in our stroma cell line as well as in bone marrow stroma from various mouse strains. Stimulation of lymphoma cells with recombinant Shh or Ihh protein could overcome apoptosis induction induced by removal of the stromal layer, indicating Ihh as a mediator between stroma and lymphoma cells. Injection of luciferazed lymphoma cells into C57Bl6 mice induced lymphoma formation within 2–6 weeks, as shown by Xenogen luciferase imaging. Subcutaneous treatment of lymphoma injected mice with Cyclopamine could inhibit lymphoma formation and even reduce lymphoma mass in mice with already fully developed lymphomas, indicating stroma-lymphoma interaction through hedgehog signaling as an important survival mechanism for lymphoma cells also in vivo. Our data demonstrates that proliferation and survival of a majority of B-cell lymphomas and plasmacytomas is dependent on intact Hh signaling in vitro and in vivo, suggesting disruption of this pathway as a novel approach in lymphoma therapy.

Description: Introduction The emergence of kinase inhibitors like Ibrutinib has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious problems. The three PIM kinases are involved in various important disease mechanisms in CLL, with PIM1 regulating CXCR4 surface expression impacting its interaction with the microenvironment, and PIM2/3 affecting the apoptotic machinery by regulating BAD. The Pan-PIM kinase inhibitor LGB321 (Novartis) targets all three PIM kinases and therefore affects both, CLL apoposis and its interaction with the microenvironment. Results In the study presented here, we investigated the effect of the Pan-PIM kinase inhibitor LGB321 on CLL in vitro and in vivo. LGB321 was highly effective in inducing apoptosis in primary human CLL cells, independent of risk factors or the mutation status. Apoptosis induction correlated with reduced pBAD and BAD levels. LGB321 was also effective in the presence of protective stromal cells and could completely overcome the stroma protective effects. Furthermore, we found that Pan-PIM inhibitor treatment blocked the CXCR4/CXCL12 axis by dephosphorylating the CXCR4 receptor on Ser339, by reducing total CXCR4 protein levels, and by blocking the externalization of the CXCR4 receptor. Concordantly, LGB321 blocked CXCR4 functions like migration towards a CXCL12 gradient (P

Description: The efficacy of first-line intensive chemotherapy (CX) with PBSCT and its use as subsequent treatment in relapsed lymphoma patients (pts) was analysed in the past. According to the International Prognostic Index (IPI), first-line auto-PBSCT was shown to be superior to standard-CHOP with intermediate- or high-risk lymphoma pts as well as pts with relapse. This suggests that CHOP alone can no longer be regarded as standard treatment for these pts. However, with increasing curative prospects also for older pts, CX-induced cardiotoxicity (CT), eventually leading to acute or chronic heart failure (HF), becomes a relevant factor on morbidity and mortality. The incidence of clinically manifest CT in pts undergoing PBSCT has been estimated between 5–10%. However, in the presence of multiple complex medical problems with several co-existing pathologies, it is often difficult to diagnose HF. BNP is a hormone mostly secreted from the heart ventricles which has recently gained popularity for the following reasons: It is 1. related to HF severity and clinical status, 2. increased as left ventricular function deteriorates due to wall stretch and tension, 3. strongly associated with prognosis across the whole spectrum of HF and 4. a powerful independent predictor of mortality and morbidity in the long-term follow-up of HF cohorts. Here, we determined cardiac markers in malignant lymphoma pts to determine PBSCT-induced CT. For cardiac assessment, BNP and Troponin T (TrT) concentrations were measured before (day [d]0) and after PBSCT (d30, 3months [m], 1year [y]). Concomitant recordings of ECG, echocardiography and clinical history were carried out. So far, 39 consecutive pts (m:f=23:16) with a median age of 55 (19–70) y have been enrolled. 23 pts had high-grade NHL, 10 pts had indolent-NHL and 6 pts had Hodgkin’s disease. Most pts had stage III or IV disease (90%). Their median IPI was 4. First-line PBSCT vs. PBSCT following relapse was performed in 22 vs. 17 pts, respectively. All pts had received standard-dose CX prior to PBSCT. The used mobilization CX were VCP-E with first-line PBSCT or DHAP following relapse achieving CR, PR or SD prior to PBSCT in 10, 13 and 9 pts, respectively. All pts received high-dose CX with BEAM, retransfusing median CD34+ cells of 4.5x10e6/kg. Treatment related mortality was 0%. Currently, 35 pts are alive. Cardiac events (CE) occurring before PBSCT were coronary heart disease in 4 pts and prior myocardial infarction in 3 pts. After PBSCT, 10 pts without prior cardiac history developed mainly arrhythmias. BNP proofed to be a significantly more sensitive marker than TrT, showing elevated levels in 69% of pts at any time post PBSCT. In comparison, TrT was never elevated. Median BNP levels on d0, d30, 3m, and 1y after PBSCT were 142, 224, 228 and 207pg/ml (normal 55y (228pg/ml) compared to pts

Description: Abstract 1310 The spleen tyrosin kinase (SYK) was shown to be overexpressed in various human hematologic malignancies like B-cell lymphomas and acute myeloid leukemias. There are two known translocations involving the SYK kinase: ITK-SYK and TEL-SYK. ITK-SYK was identified in unspecified peripheral T-cell lymphomas, whereas the TEL-SYK fusion had been discovered in a patient with myelodysplastic syndrome (MDS). Methods: All SYK containing oncogenes, ITK-SYK, TEL-SYK and SYK wt, were cloned into a pMSCV-IRES-GFP retroviral vector. All oncogenes and a GFP control vector were overexpressed in bone marrow of 5-flurouracil-pretreated mice and oncogene containing bone marrow was transplanted into irradiated recipient mice. Disease development was monitored over 1 year. Additionally the three SYK oncogenes were overexpressed in a myeloid (32D), B-lymphoid (BaF3) and T-cell lineage (Jurkat) to monitor cell context specific differences in cell signaling. Results: Here, we demonstrate that the overexpression of 3 SYK containing oncogenes, SYK wt, TEL-SYK and ITK-SYK in a murine bone marrow transplantation model induces the development of 3 different hematologic (pre)diseases. While ITK-SYK exclusively induced T-cell lymphomas in mice, we found that the TEL-SYK fusion induced an MDS/MPS overlapsyndrom. The mice developed an anemia and thrombopenia with signs of dysplasia of megakaryocytes and the erythroid lineage in the bone marrow and spleens. In addition those mice showed a strong expansion of the myeloid lineage, with leucocytosis in the peripheral blood mainly consisting of differentiated neutrophils. The bone marrow shows expansion of differentiated myeloid cells, but also myeloid progenitors and expansion of HSCs. Interestingly, also SYK wt induced a relative expansion of the myeloid compartment without inducing a leucocytosis or a lethal disease indicating a premalignant myeloproliferative state. Only TEL-SYK was able to induce IL-3 independent growth of BaF3- and 32D cells and could activate STAT5, STAT6, ERK, PLCγ2 and SLP76 in those cell lines. SYK and ITK-SYK were able to activate PLCγ2 and SLP76, but no STAT transcription factors. STAT5 activation by TEL-SYK was detectable also in vivo, which might indicate STAT5 as the major driver of TEL-SYK induced transformation. Our results demonstrate, that the various SYK oncogenes can induce different hematologic diseases in mice and reveales TEL-SYK as the first oncogene inducing an MDS/MPS overlap syndrome in mice. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3864 Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B lymphocytes. For decades, nucleoside analogs, alkylating agents, and immunotherapeutics have remained the mainstay in treating this disease. Despite major advances in this field, CLL remains incurable with standard therapy. In recent years, preclinical and early clinical data on the use of kinase inhibitors have sparked new hope in the treatment of CLL. The multikinase inhibitor sorafenib, targeting RAF, platelet-derived growth factor receptor (PDGFR), KIT, FMS-like tyrosine kinase 3 (FLT3), and vascular endothelial growth factor receptor (VEGFR), has been approved for the treatment of renal cell carcinoma and hepatocellular carcinoma. Recent studies suggested that CLL cells might be also susceptible to this compound, however the precise mode of action in CLL cells remains elusive. In this study, we identified the Insulin-like growth factor receptor-I (IGF1R) pathway as novel target of sorafenib inducing cell death in CLL cells. Treatment with 10 μM of sorafenib significantly increased apoptosis in primary CLL cells as determined by AnnexinV/PI staining via flow cytometry. Commensurate with its RAF inhibiting properties, the apoptotic effect of sorafenib was accompanied with ERK pathway inhibition. Moreover, sorafenib treatment decreased phosphorylation of SRC and AKT, molecules implicated with IGF1R and insulin receptor (IR) signaling. Interestingly, the latter were strongly expressed in primary CLL cells compared with healthy B cells. Similar to sorafenib, 24 hour treatment of CLL cells with the three structurally distinct IGF1R inhibitors Picropodophyllin, AG1024, and Linsitinib significantly increased apoptosis compared with vehicle control resulting in decreased phophorylation of MEK, ERK, SRC, and AKT. Sorafenib and the IGF1R inhibitor AG1024 also downregulated the expression of IGF1R on CLL cells but not on healthy B cells. To test whether sorafenib modulates IGF-1 binding and thereby influences the IGF1R activation, we biotinylated recombinant IGF-1 and tested its binding to the IGF1R via flow cytometry. We observed a reduced IGF-1 binding after sorafenib treatment. IGF-1 binding after treatment with different IGF1R inhibitors was performed as an internal control. In order to further establish the functional relevance of IGF1R expression in CLL, we performed IGF1R specific and non-silencing siRNA experiments in primary CLL cells. In line with our previous results, IGF1R knockdown resulted in a significant decrease of cell viability and in downregulation of RAF-1 expression, and MEK, ERK, SRC, and AKT phosphorylation. The stromal microenvironment protects CLL cells from spontaneous and drug-induced apoptosis. Sorafenib, AG1024, and Picropodophyllin counteracted the protective effect of microenvironmental factors simulated by the presence of the murine stromal cell line M210B4, the chemokine CXCL12, and the integrin CD49d. Finally, we used the Eμ-Tcl1 transgenic mouse model to validate these results in vivo. Male and female mice (n=8) were treated with 25 mg/kg of the IGF1R inhibitor Linsitinib per oral gavage for 7 days and the amount of CD5/CD19 positive cells was determined flow cytometrically at different time points. We observed a reduction of CD5/CD19-positive cells by 26,1% and 23,2% after 4 and 8 days of treatment, respectively. Our results provide a novel mechanism of action of the multikinase inhibitor sofarenib in CLL cells by blocking IGF1R mediated signaling. IGF1R inhibition by itself induced apoptosis in CLL cells in vitro and in vivo, thus identifying IGF1R as promising target for therapeutic approaches and proposing IGF1R inhibitors for clinical assessment in the therapy of CLL. Disclosures: No relevant conflicts of interest to declare.