Bone Marrow Stromal Cells Drive Key Hallmarks of B Cell Malignancies.

All B cell leukaemias and a substantial fraction of lymphomas display a natural niche residency in the bone marrow. While the bone marrow compartment may only be one of several sites of disease manifestations, the strong clinical significance of minimal residual disease (MRD) in the bone marrow strongly suggests that privileged niches exist in this anatomical site favouring central elements of malignant transformation. Here, the co-existence of two hierarchical systems, originating from haematopoietic and mesenchymal stem cells, has extensively been characterised with regard to regulation of the former (blood production) by the latter. How these two systems cooperate under pathological conditions is far less understood and is the focus of many current investigations. More recent single-cell sequencing techniques have now identified an unappreciated cellular heterogeneity of the bone marrow microenvironment. How each of these cell subtypes interact with each other and regulate normal and malignant haematopoiesis remains to be investigated. Here we review the evidences of how bone marrow stroma cells and malignant B cells reciprocally interact. Evidently from published data, these cell-cell interactions induce profound changes in signalling, gene expression and metabolic adaptations. While the past research has largely focussed on understanding changes imposed by stroma- on tumour cells, it is now clear that tumour-cell contact also has fundamental ramifications for the biology of stroma cells. Their careful characterisations are not only interesting from a scientific biological viewpoint but also relevant to clinical practice: Since tumour cells heavily depend on stroma cells for cell survival, proliferation and dissemination, interference with bone marrow stroma-tumour interactions bear therapeutic potential. The molecular characterisation of tumour-stroma interactions can identify new vulnerabilities, which could be therapeutically exploited

ZAP-70 Shapes the Immune Microenvironment in B Cell Malignancies.

Zeta-chain-associated protein kinase-70 (ZAP-70) is a tyrosine kinase mainly expressed in T cells, NK cells and a subset of B cells. Primarily it functions in T cell receptor (TCR) activation through its tyrosine kinase activity. Aberrant expression of ZAP-70 has been evidenced in different B cell malignancies, with high expression of ZAP-70 in a subset of patients with Chronic Lymphocytic Leukemia (CLL), associating with unfavorable disease outcomes. Previous studies to understand the mechanisms underlying this correlation have been focused on tumor intrinsic mechanisms, including the activation of B cell receptor (BCR) signaling. Recent evidence also suggests that ZAP-70, intrinsically expressed in tumor cells, can modulate the cross-talk between malignant B cells and the immune environment, implying a more complex role of ZAP-70 in the pathogenesis of B cell malignancies. Meanwhile, the indispensible roles of ZAP-70 in T cell and NK cell activation also demonstrate that the autologous expression of ZAP-70 in the immune environment can be a central target in modulation of tumor immunity. Here we review the evidences of the link between ZAP-70 and tumor immunology in the microenvironment in B cell malignancies. Considering an emerging role of immunotherapies in treating these conditions, understanding the distinct molecular functions of ZAP-70 in a broader cellular context could ultimately benefit patient care

Chronic Lymphocytic Leukemia increases the pool of peripheral blood hematopoietic stem cells and skews differentiation

Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy invariably infiltrating the bone marrow. Although treatment options for patients with advanced disease have significantly improved in the past years, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary bone marrow failure. The underlying mechanisms impairing normal hematopoiesis in patients with CLL are poorly defined.We would like to express our deepest gratitude to patients who donated blood for this research. Samples were obtained with assistance from the Cambridge Blood and Stem Cell Biobank, funded by the Cambridge Cancer Centre and Cambridge Stem Cell Institute. This work was funded by the Cancer Research UK (CRUK; C49940/A17480). I.R. is a senior CRUK fellow. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA and Royal Society. Research in I.R. and E.L. laboratories is supported by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute

Lck is a relevant target in chronic lymphocytic leukaemia cells whose expression variance is unrelated to disease outcome.

Pathogenesis of chronic lymphocytic leukaemia (CLL) is contingent upon antigen receptor (BCR) expressed by malignant cells of this disease. Studies on somatic hypermutation of the antigen binding region, receptor expression levels and signal capacity have all linked BCR on CLL cells to disease prognosis. Our previous work showed that the src-family kinase Lck is a targetable mediator of BCR signalling in CLL cells, and that variance in Lck expression associated with ability of BCR to induce signal upon engagement. This latter finding makes Lck similar to ZAP70, another T-cell kinase whose aberrant expression in CLL cells also associates with BCR signalling capacity, but also different because ZAP70 is not easily pharmacologically targetable. Here we describe a robust method of measuring Lck expression in CLL cells using flow cytometry. However, unlike ZAP70 whose expression in CLL cells predicts prognosis, we find Lck expression and disease outcome in CLL are unrelated despite observations that its inhibition produces effects that biologically resemble the egress phenotype taken on by CLL cells treated with idelalisib. Taken together, our findings provide insight into the pathobiology of CLL to suggest a more complex relationship between expression of molecules within the BCR signalling pathway and disease outcome

Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia.

Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5'-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy

Negative feedback regulation of MAPK signaling is an important driver of chronic lymphocytic leukemia progression

Despite available targeted treatments for the disease, drug-resistant chronic lymphocytic leukemia (CLL) poses a clinical challenge. The objective of this study is to examine whether the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate mitogen-activated protein kinases (MAPKs) and thus counterbalance excessive MAPK activity. We show that high expression of DUSP6 in CLL correlates with poor clinical prognosis. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small-molecule inhibitor reduces CLL cell survival in vitro and in vivo. Using global phospho-proteome approaches, we observe acute activation of MAPK signaling by DUSP1/6 inhibition. This promotes accumulation of mitochondrial reactive oxygen species and, thereby, DNA damage and apoptotic cell death in CLL cells. Finally, we observe that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising treatment concept to eliminate drug-resistant CLL cells

Stromal cell protein kinase C-β inhibition enhances chemosensitivity in B cell malignancies and overcomes drug resistance.

Overcoming drug resistance remains a key challenge to cure patients with acute and chronic B cell malignancies. Here, we describe a stromal cell-autonomous signaling pathway, which contributes to drug resistance of malignant B cells. We show that protein kinase C (PKC)-β-dependent signals from bone marrow-derived stromal cells markedly decrease the efficacy of cytotoxic therapies. Conversely, small-molecule PKC-β inhibitors antagonize prosurvival signals from stromal cells and sensitize tumor cells to targeted and nontargeted chemotherapy, resulting in enhanced cytotoxicity and prolonged survival in vivo. Mechanistically, stromal PKC-β controls the expression of adhesion and matrix proteins, required for activation of phosphoinositide 3-kinases (PI3Ks) and the extracellular signal-regulated kinase (ERK)-mediated stabilization of B cell lymphoma-extra large (BCL-XL) in tumor cells. Central to the stroma-mediated drug resistance is the PKC-β-dependent activation of transcription factor EB, regulating lysosome biogenesis and plasma membrane integrity. Stroma-directed therapies, enabled by direct inhibition of PKC-β, enhance the effectiveness of many antileukemic therapies.This work was funded by Cancer Research UK (CRUK; C49940/A17480). I.R. is a senior CRUK fellow. M.S.S is supported by the DFG through SCHM2440/7-1 and CRC1243 (A12). L.G. & O.W. received funding from CWCUK (grant 14-169) and GOSHCC (grant V2617). A.E. receives research grants from the Austrian Science Fund (FWF; Transcan I2795-B28 to A.E. (FIRE-CLL), DACH grants I3282-B26 and I1299-B21 (FOR2036) and a grant from the Paracelsus Medical University (PMU Grant E-13/18/091-EGF). S.S. receives funding from the DFG (SFB1074 , project B1), relevant to this work

Investigator choice of standard therapy versus sequential novel therapy arms in the treatment of relapsed follicular lymphoma (REFRACT): study protocol for a multi-centre, open-label, randomised, phase II platform trial

BackgroundRelapsed or refractory follicular lymphoma (rrFL) is an incurable disease associated with shorter remissions and survival after each line of standard therapy. Many promising novel, chemotherapy-free therapies are in development, but few are licensed as their role in current treatment pathways is poorly defined. MethodsThe REFRACT trial is an investigator-initiated, UK National Cancer Research Institute, open-label, multi-centre, randomised phase II platform trial aimed at accelerating clinical development of novel therapies by addressing evidence gaps. The first of the three sequential novel therapy arms is epcoritamab plus lenalidomide, to be compared with investigator choice standard therapy (ICT). Patients aged 18 years or older with biopsy proven relapsed or refractory CD20 positive, grade 1-3a follicular lymphoma and assessable disease by PET-CT are eligible. The primary outcome is complete metabolic response by PET-CT at 24 weeks using the Deauville 5-point scale and Lugano 2014 criteria. Secondary outcomes include overall metabolic response, progression-free survival, overall survival, duration of response, and quality of life assessed by EQ-5D-5 L and FACT-Lym. The trial employs an innovative Bayesian design with a target sample size of 284 patients: 95 in the ICT arm and 189 in the novel therapy arms. Discussion:Whilst there are many promising novel drugs in early clinical development for rrFL, understanding the relative efficacy and safety of these agents, and their place in modern treatment pathways, is limited by a lack of randomised trials and dearth of published outcomes for standard regimens to act as historic controls. Therefore, the aim of REFRACT is to provide an efficient platform to evaluate novel agents against standard therapies for rrFL. The adaptive Bayesian power prior methodology design will minimise patient numbers and accelerate trial delivery. Trial registration: ClinicalTrials.gov: NCT05848765; 08-May-2023. EudraCT: 2022-000677-75; 10-Feb-2022

Viral transduction of primary human lymphoma B cells reveals mechanisms of NOTCH-mediated immune escape

Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo