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The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double–cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Novel targeted therapies have substantially improved the prognosis of patients with B cell malignancies. However, a substantial fraction of patients still relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/ lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance (EMDR)). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumour cell adhesion enabling the physical displacement from protective niches (e.g. BCR-inhibitors). In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we recently characterised the molecular mechanisms underlying tumor-stroma interactions in B cell malignancies and identified a protein kinase C-β (PKC-β) as an essential kinase, required for activation of NF-κB in mesenchymal stromal cells (Lutzny et al Cancer Cell 2013). The dependency on stroma PKC-β was uniformly found for acute (ALL) and chronic (CLL, MCL) B cell malignancies. Importantly, our data further demonstrate that targeting stroma PKC-β is of key importance for multi-drug resistance of malignant B cells and can be used for therapeutic interventions (Park et al Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B cell malignancies. We identified that stroma PKC-β drives a transcriptional program in tumor cells, dependent on the activation of TGF-β and BMP-signaling, which ultimately leads to the stabilisation of BCL-XL. Our data show that BCL-XL expression in tumor cells is associated with SMAD1-induction by cytotoxic therapies, which simultaneously suppress SMAD4 expression. Importantly, SMAD1 expression was strictly dependent on stromal PKC-β activity. Antagonizing stroma signals with TGF-β inhibitors inhibits SMAD1 induction, abrogates the up-regulation of BCL-XL and overcomes stroma-dependent resistance to Venetoclax and conventional chemotherapy. The TGF-β pathway operates in parallel to the activation of the transcription factor EB (TFEB) as a down-stream target of PKC-β. Interference with these signaling pathways impairs plasma membrane integrity of stromal cells by down-regulation of numerous adhesion and signaling molecules, such as ADAM17, required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-β for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EμTCL-1 driven B cell tumors and treated with Venetoclax in combination with or without PKC-β inhibitors. Combined treatment significantly prolonged survival, based on PKC-β mediated impairment of EMDR. Similarly, concurrent treatment of PKC-β inhibitors with chemotherapy also improved survival in an ALL-PDx model Our data demonstrate that mitigating EMDR with small molecule inhibitors of PKC-β or TGF-β signalling enhance the effectiveness of both targeted and non-targeted chemotherapies and moreover, has the ability to overcome Venetoclax resistance in B cell malignancies. Clinical trials with repurposed drugs inhibiting the here described pathways mediating EMDR are in planning. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Midostaurin as inhibitor of stroma PKC-β

The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.

The recent clinical success with small molecule inhibitors targeting the B cell receptor (BCR) pathway provides unambiguous evidence that signals engaging the BCR play a key role in the pathogenesis of CLL. The binding of CLL cells to a proposed auto-antigen has encouraged many researchers to identify these antigens. This has more recently been challenged by the identification of an internal epitope within the BCR leading to a cell autonomous, antigen-independent activation of the BCR. Overall, the response of CLL cells to a continuous activation of the BCR is anergy. Notably, with respect to different subgroup of patients, M-CLL/ZAP70 negative cases appear to be more "locked" in an anergic state than UM/ZAP70 positive cases. Since investigations in non-malignant B cells suggest that innate signaling-pathways integrate into the BCR-signaling cascade to overcome anergy, we investigated the responses to TLR9 ligation in 57 purified CLL patient samples with regard to the expression of ZAP70, which is considered to amplify signals funneled through the BCR: Here we report that ZAP70 positive CLL shows an increased proliferative response (p=0.005) and enhanced survival (p=0.001) upon TLR9 stimulation when compared to ZAP70 negative CLL. Notably, compared to untreated controls, TLR9 ligation induced apoptosis in ZAP70 negative clones, as opposed to ZAP70 positive cells, in which TLR9 agonists inhibit cell death. TLR9-conditioned media from ZAP70 positive cells completely inhibited the pro-apoptotic effect of TLR9 on ZAP70 negative cells, indicating that soluble factors protect cells from TLR9 induced cell death. Analyzing the expression and modulation of BH3 proteins in TLR9 activated CLL cells, we observed a loss of the pro-apoptotic protein BIM in ZAP70 positive cells, strongly correlating with prolonged survival. Notably, increased BIM expression upon TLR9 ligation was observed in ZAP70 negative cells. In non-malignant B cells BIM is degraded upon activation of the BCR, but not due to the ligation of TLR9. Therefore, we hypothesized that BCR-mediated signals are involved in the TLR9 response of ZAP70 positive CLL. This hypothesis is further supported by the finding that TLR9 mediated loss of BIM in ZAP70 positive CLL was inhibited by the highly specific Syk inhibitor P505-15. Accordingly, TLR9 mediated survival of CLL cells was decreased by P505-15. Since the anti-apoptotic effect of TLR9 stimulation is mediated by soluble factors, we analyzed the secretom of TLR9-conditioned media from CLL samples by mass spectrometry. Surprisingly, we found secreted IgM in conditioned media from TLR9 stimulated, ZAP70 positive, but not negative CLL. TLR9 induced secretion of IgM could also be antagonized with P505-15, further supporting our finding that TLR9 ligation impinges on BCR signaling in ZAP70 positive CLL. In addition, TLR9 conditioned media depleted of soluble IgM, failed to protect cells from apoptosis, indicating that IgM is essential to rescue TLR9 stimulated cells from cell death. In summary, our data strongly indicate that -in ZAP70 positive CLL- TLR9 ligation induces the secretion of IgM and down-regulation of BIM, both features of BCR activation. We propose the idea that the soluble IgM binds to the surface BCR through an internal epitope interaction, engaging a positive feed-back loop and further promoting the survival of ZAP70 positive CLL. Thus, ZAP70 may operate as an amplifier for TLR9 mediated activation of the BCR signaling-cascade. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other.

Heterotypic interactions between bone marrow stromal cells (BMSCs) and malignant B cells contribute to apoptosis-resistance of tumour cells, based on the provision of anti-apoptotic factors by stromal cells. For this reason, interference with the microenvironment may offer an alternative approach to chemotherapy to target B cell lymphomas/ leukaemias. However, the success of such treatments critically relies on specific targets expressed in the lymphoma microenvironment. We have previously reported that monoclonal B cells from patients with CLL, MCL und ALL activate and reprogram BMSCs. This activation of stromal cells is an essential prerequisite for microenvironment-mediated survival of malignant B cells and requires the induction and activation of protein-kinase C-β in stromal cells in vitro and in vivo. This is underscored by a complete resistance of PKC-β knockout mice to adoptively transferred B cell lymphomas from TCL1 transgenic mice (Lutzny et al. Cancer Cell. 2013 Jan 14; 23(1):77-92.). Analyses of primary CLL cells co-cultured on human or mouse BMSCs indicate that stromal cells protect malignant B cells from apoptosis primarily by enhancing the expression of the anti-apoptotic proteins Mcl1, XIAP, BclXL and Bcl2A1. Knockdown of Mcl1 expressed in CLL cells further demonstrates that its expression is crucial for stroma-mediated survival even in the presence of BMSCs. Since BMSC-mediated survival and propagation of CLL depends on the kinase activity of PKC-β, we hypothesized that pharmacological inhibition of the activation of stromal cells using small molecule inhibitors against PKC-β may display anti-leukemic effects. Here we report that the PKC-β inhibitor enzastaurin enhances the cytotoxic effects of standard chemotherapeutic drugs and of the BCL2-inhibitor ABT737. Dose-response analyses indicate that enzastaurin potentiates the cytotoxic effects of ABT737 in a synergistic manner, rapidly causing caspase-mediated apoptosis of malignant B cells. This drug-sensitising effect of enzastaurin is mediated by inhibition of PKC-β induced and expressed in activated stromal cells and not related to a direct cytotoxic effect on malignant B cells: PKC-β deficient BMSCs fail to maintain high expression levels of the anti-apoptotic proteins Mcl1, XIAP and Bcl2A1 in CLL cells, which show a significantly enhanced sensitivity to ABT737 compared to CLL cells cultured on PKC-β proficient stromal cells. These data provide evidence that PKC-β inhibitors can therapeutically be used to abolish microenvironment-mediated survival of malignant B cells. In order to assess whether drug combinations of enzastaurin and BCL2 inhibitors demonstrate synergistic anti-lymphoma effects in vivo, we adoptively transferred malignant B cells from diseased TCL1 mice into wild-type recipient mice. Tumour-bearing mice were treated with enzastaurin, ABT199 or a combination of both drugs. Response to treatment, assessed by PET-CT scan, indicates that the combination of enzastaurin and ABT199 is superior to single agent treatment. These data indicate that malignant B cells can be sensitized to cytotoxic drugs by inhibiting the tumour-promoting effects of the lymphoma microenvironment with PKC-β inhibitors. Our preclinical in vitro and in vivo experiments justify testing this drug combination as a new approach to leukaemia/ lymphoma therapy in a phase I/II clinical trial. Disclosures: No relevant conflicts of interest to declare.

Abstract 314 Defects in the apoptosis program are a hallmark of chronic lymphocytic leukemia (CLL), characterized by high expression levels of bcl2 and Mcl1. Notably, this de-regulation of anti-apoptotic proteins is not sufficient to maintain long-term survival of CLL cells, which remain highly dependent on pro-survival factors provided by the leukemia-microenvironment. Bone marrow stromal cells (BMSCs) play an important role for microenvironment mediated survival of CLL cells, based on the provision of soluble and membrane-bound factors. The stroma-CLL interactions not only protect CLL cells from spontaneous, but also from drug-induced apoptosis, clinically recognized as minimal-residual disease. Therefore, understanding the molecular mechanisms of CLL-stroma interactions may offer new therapeutic options and help in eradicating CLL cells from the bone-marrow niche. Here we describe that monoclonal B-cells from CLL patients impose morphological and genetic changes in stromal cells, which become reminiscent of cancer-associated fibroblast (CAF). Comparative gene expression profiles indicate that contact with primary CLL cells induce the expression of pro-inflammatory genes in stromal cells. Further characterization of the underlying signaling pathways activated in stromal cells revealed that CLL cells induce the expression of protein-kinase C-β in BMSCs. Blocking the up-regulation of PKC-β by siRNA abrogated the pro-survival effects of stromal cells on CLL cells. Furthermore, following induction of PKC-β, BMSCs activate NF-kappaB through a Bcl10-independent, but NEMO/IKKgamma-dependent pathway. Gene expression profiling of NEMO-proficient and deficient BMSCs indicated that NF-kappaB regulates the expression of pro-inflammatory cytokines and adhesion molecules by stromal cells, required to promote survival of CLL. Interference with the NF-kappaB activation in BMSCs abrogated the pro-survival effects of stromal cells on CLL, similar to PKC-β deficient stromal cells. To demonstrate that this pathway is also important in vivo, Tcl1-CLL was transplanted into syngeneic PKC-β knock-out and wild-type mice. Notably, all PKC-β wild-type mice died of a CLL-like disease, whereas PKC-β kock-out animals were entirely resistant to CLL transplants. Immunofluorescence staining of PKC-β in bone marrow trephine biopsies indicated that this pathway is also activated in mesenchymal stromal cells of CLL patients. Importantly, our data provide further evidence that the PKC-β – NF-kappaB pathway is also activated in stomal cells by monoclonal B-cells from ALL and mantle-cell lymphoma (MCL) patients. Conclusively, we describe a novel survival signaling pathway activated by monoclonal B-cells in BMSCs. Interference with the PKC-β-NF-kappaB pathway activated in the leukemia/lymphoma microenvironment may offer new therapeutic options to fully eradicate malignant B-cells from bone-marrow niches. Disclosures: No relevant conflicts of interest to declare.

Abstract 2735 Poster Board II-711 Patients with hematologic malignancies have a high risk of developing invasive fungal infections. The higher risk is attributed to host defense impairment due to intensive cytotoxic chemotherapies, hematopoetic stem cell transplantation, and immunosuppressive agents. As early treatment initiation in patients with invasive fungal infections has a profound impact on mortality rates, different antifungal treatment strategies like prophylaxis, empirical, pre-emptive and targeted treatment after proven fungal infection have been developed. Azoles are the most broadly used antifungal drugs inhibiting CYP51. They play a pivotal role in the treatment and prophylaxis of systemic and dermal mycoses. Azoles inhibit the sterol 14a-demethylase activity and block sterol biosynthesis, which is lethal in unicellular organisms. However, in mammalian cells, it has been shown that they lower endogenous cholesterol production. As lipid rafts consist of sphingolipids and cholesterol and lipid rafts have been demonstrated to be crucial for rituximab induced cell death, we asked whether rituximab exhibits its full anti-lymphoma effect in the presence of azoles. We now demonstrate that antifungal treatment with itraconazole impairs rituximab's anti-lymphoma effect both in vitro and in vivo. Using a mouse xenograft model, no tumor growth was observed in mice treated with rituximab. In contrast, in the presence of itraconazole there was tumor growth, indicating that the concentration was sufficient to antagonize rituximab′s anti-lymphoma effect. On the molecular level, CD20 raft recruitment was inhibited in the presence of itraconazole. Furthermore, the translocation of CD20 into lipid rafts has been shown to be crucial for calcium influx and apoptosis. Therefore, we tested the rituximab induced calcium flux in the absence and presence of itraconazole. Indeed, calcium flux was almost completely abolished in the presence of itraconazole. The fact that antibody treatment failure in the presence of itraconazole only affects targeted therapy against raft associated molecules i.e. CD20 and CD52 but not the non raft associated molecule CD33 further supports our proposed mechanism of action. Together, our data suggest that concomitant treatment of itraconazole and rituximab attenuates rituximab's anti-lymphoma effect. As chemotherapy by itself has high response rates in lymphoma, the loss of rituximab mediated cell death in the presence of itraconacole may be missed in our daily routine. Disclosures: No relevant conflicts of interest to declare.

Abstract 2354 Poster Board II-331 A defect in the programmed cell death, apoptosis, is implemented in the pathogenesis of CLL. About ten years ago, it became evident that patients with CLL can be divided into those with an indolent course of the leukaemia and those which suffer from a more aggressive disease, typically requiring frequent chemotherapy and ultimately develop a chemotherapy-refractory state. The latter group of patients aberrantly express the T-cell associated protein ZAP-70. The object of this study was to identify the molecular differences underlying the pathogenesis of these two CLL subgroups. To study differences in the apoptotic program we used primary CLL cells derived from untreated ZAP-70 negative and positive patients. Here we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor (BCR) and recruits protein kinase C-beta (PKC-beta) into lipid raft domains only in patients with an aggressive variant of the disease. Subsequently, PKC-beta is activated and shuttles from the plasma membrane into the mitochondria. By using co-immunoprecipitation experiments and PKC-beta specific small molecule inhibitors we unravel that the anti-apoptotic protein Bcl-2 and its antagonistic BH3-protein Bim are putative substrates for PKC-beta. PKC-beta mediated phosphorylation of Bcl-2 augments its anti-apoptotic function by increasing its ability to sequester more pro-apoptotic Bim. In addition, the phosphorylation of Bim by PKC-beta leads to its proteasomal degradation. Therefore, high levels of phospho-Bcl-2 and low levels of Bim are a hallmark of ZAP-70 positive, aggressive CLL. Importantly, posttranscriptional modifications of Bcl-2 seem to outweigh the absolute expression of Bcl-2 with respect to the suppression of apoptosis. We demonstrate that these cells are strongly protected from chemotherapy-induced cytotoxic stress. Our data indicate that the constitutive activation of PKC-beta is directly involved in the apoptotic defect in ZAP-70 positive CLL. We finally show that targeting PKC-beta is an attractive approach to the treatment of CLL patients. Enzastaurin is a PKC-beta specific inhibitor and currently tested in clinical phase I/II trials for cancer patients. Our data demonstrate that this compound is highly active in CLL cells and augments the cytotoxic effects of standard chemotherapeutic drugs. Our results provide evidence that the constitutive activation of PKC-beta is directly implicated in the pathogenesis of aggressive CLL by altering the function of the apoptosis-regulating proteins Bcl-2 and Bim. These changes confer cells to a more anti-apoptotic state with aggressiveness of the disease. Targeting PKC-beta with small-molecule inhibitors like Enzastaurin might offer a new therapeutic strategy to control or even cure CLL. Disclosures: No relevant conflicts of interest to declare.

Purpose: We have previously shown that PKC delta is constitutively activated in B-CLL cells. In the present study, we have analyzed the mechanism of apotosis induction as well as the effets of PKC delta in the presence of survival signal and chemotherapeutic agents. Methods: The mitochondrial membrane potential, presence of active caspase 3, conformational status of Bax and apoptosis assays (Annexin V stain, Tunel assay) were performed using flow cytometric analysis. Expression of BCL-2 family proteins and XIAP as well as processing of caspase 8 and 9 was revealed in western blot experiments. Results: Rottlerin at 5μM (which is not toxic for normal lymphocytes) induced apoptosis in a large amount of B-CLL samples. Rottlerin was equally effective in zap70 positive- and negative samples. Rottlerin induced apoptosis was in part caspase dependent and involved processing of caspase 3, 8 and 9. Expression of antiapoptotic proteins MCL-1 and XIAP was reduced in Rottlerin treated cells and BAX expression increased and Bax conformation changed to its proapoptotic form.IN contrast, expression of bcl-2 was not changed. B-CL cells are likely to receive survival signals from the microenvironment and might be rescued from cell death induced by chemotherapeutic agents. Rottlerin was very effective in inhibiting survival signals like IL-4 or stromal cell contact. In addition, treatment with Rottlerin enhanced the cytotoxicity of the chemotherapeutic agents Fludarabine, Vncristine and Daunorubicine. Conclusion: Inhibition of PKC delta might be a powerful treatment option for B-CLL given its potential to induce apoptosis, inhibit antiapoptotic survival signals and augment the cytotoxicity of chemotherapeutic agents.