Post-transplant-cyclophosphamide and short-term Everolimus as graft-versus-host-prophylaxis in patients with relapsed/refractory lymphoma and myeloma-Final results of the phase II OCTET-EVER trial.

BACKGROUND: Conditioning regimens and the choice of immunosuppression have substantial impact on immune reconstitution after allogeneic hematopoietic stem cell transplantation (aHSCT). The pivotal mechanism to maintain remission is the induction of the graft-versus-tumor effect. Relapse as well as graft versus host disease remain common. Classic immunosuppressive strategies implementing calcineurin inhibitors (CNI) have significant toxicities, hamper the immune recovery, and reduce the anti-cancer immune response. METHODS: We designed a phase II clinical trial for patients with relapsed and refractory lymphoid malignancies undergoing aHSCT using a CNI-free approach consisting of post-transplant cyclophosphamide (PTCy) and short-term Everolimus after reduced-intensity conditioning and matched peripheral blood stem cell transplantation. The results of the 19 planned patients are presented. Primary endpoint is the cumulative incidence and severity of acute GvHD. RESULTS: Overall incidence of acute GvHD was 53% with no grade III or IV. Cumulative incidence of NRM at 1, 2, and 4 years was 11%, 11%, and 16%, respectively, with a median follow-up of 43 months. Cumulative incidence of relapse was 32%, 32%, and 42% at 1, 2, and 4 years after transplant, respectively. Four out of six early relapses were multiple myeloma patients. Overall survival was 79%, 74%, and 62% at 1, 2, and 4 years. GvHD-relapse-free-survival was 47% after 3 years. CONCLUSIONS: Using PTCy and short-term Everolimus is safe with low rates of aGvHD and no severe aGvHD or cGvHD translating into a low rate of non-relapse mortality. Our results in this difficult to treat patient population are encouraging and warrant further studies.

Transcriptomic profiles and 5-year results from the randomized CLL14 study of venetoclax plus obinutuzumab versus chlorambucil plus obinutuzumab in chronic lymphocytic leukemia.

Data on long-term outcomes and biological drivers associated with depth of remission after BCL2 inhibition by venetoclax in the treatment of chronic lymphocytic leukemia (CLL) are limited. In this open-label parallel-group phase-3 study, 432 patients with previously untreated CLL were randomized (1:1) to receive either 1-year venetoclax-obinutuzumab (Ven-Obi, 216 patients) or chlorambucil-Obi (Clb-Obi, 216 patients) therapy (NCT02242942). The primary endpoint was investigator-assessed progression-free survival (PFS); secondary endpoints included minimal residual disease (MRD) and overall survival. RNA sequencing of CD19-enriched blood was conducted for exploratory post-hoc analyses. After a median follow-up of 65.4 months, PFS is significantly superior for Ven-Obi compared to Clb-Obi (Hazard ratio [HR] 0.35 [95% CI 0.26-0.46], p < 0.0001). At 5 years after randomization, the estimated PFS rate is 62.6% after Ven-Obi and 27.0% after Clb-Obi. In both arms, MRD status at the end of therapy is associated with longer PFS. MRD + ( ≥ 10-4) status is associated with increased expression of multi-drug resistance gene ABCB1 (MDR1), whereas MRD6 (< 10-6) is associated with BCL2L11 (BIM) expression. Inflammatory response pathways are enriched in MRD+ patient solely in the Ven-Obi arm. These data indicate sustained long-term efficacy of fixed-duration Ven-Obi in patients with previously untreated CLL. The distinct transcriptomic profile of MRD+ status suggests possible biological vulnerabilities.

BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

MARCKS affects cell motility and response to BTK inhibitors in CLL.

Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

ATM activity in T cells is critical for immune surveillance of lymphoma in vivo.

The proximal DNA damage response kinase ATM is frequently inactivated in human malignancies. Germline mutations in the ATM gene cause Ataxia-telangiectasia (A-T), characterized by cerebellar ataxia and cancer predisposition. Whether ATM deficiency impacts on tumor initiation or also on the maintenance of the malignant state is unclear. Here, we show that Atm reactivation in initially Atm-deficient B- and T cell lymphomas induces tumor regression. We further find a reduced T cell abundance in B cell lymphomas from Atm-defective mice and A-T patients. Using T cell-specific Atm-knockout models, as well as allogeneic transplantation experiments, we pinpoint impaired immune surveillance as a contributor to cancer predisposition and development. Moreover, we demonstrate that Atm-deficient T cells display impaired proliferation capacity upon stimulation, due to replication stress. Altogether, our data indicate that T cell-specific restoration of ATM activity or allogeneic hematopoietic stem cell transplantation may prevent lymphomagenesis in A-T patients.

Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia.

Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.

Chimeric antigen receptor T cells targeting Fc µ receptor selectively eliminate CLL cells while sparing healthy B cells.

Adoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)-modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged "on-target off-tumor" toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcµR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcµR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcµR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcµR CAR T cells did not attack healthy B cells. T cells with anti-FcµR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcµR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcµR CAR T cells for the treatment of CLL.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Concepts of Chronic Lymphocytic Leukemia Pathogenesis: DNA Damage Response and Tumor Microenvironment.

Pathogenesis of chronic lymphocytic leukemia (CLL) is characterized by specific genetic aberrations and alterations of cellular signaling pathways. In particular, a disturbed DNA damage response (DDR) and an activated B-cell receptor signaling pathway play a major role in promoting CLL cell survival. External stimuli are similarly essential for CLL cell survival and lead to activation of the PI3K/AKT and MAPK pathways. Activation of nuclear factor-kappa B (NFkB) influences the disturbed anti-apoptotic balance of CLL cells. Losses or disabling mutations in TP53 and ATM are frequent events in chemotherapy-naïve patients and are further enriched in chemotherapy-resistant patients. As these lesions define key regulatory elements of the DDR pathway, they also determine treatment response to genotoxic therapy. Novel therapeutic strategies therefore try to circumvent defective DDR signaling and to suppress the pro-survival stimuli received from the tumor microenvironment. With increasing knowledge on specific genetic alterations of CLL, we may be able to target CLL cells more efficiently even in the situation of mutated DDR pathways or protection by microenvironmental stimuli.

Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

miRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in CLL.

Resistance toward CD95-mediated apoptosis is a hallmark of many different malignancies, as it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are downregulated in CLL cells. Here, we identified 2 new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly overexpressed in CLL cells. APTs are the only enzymes known to promote depalmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared with normal B cells. We identified APTs to directly interact with CD95 to promote depalmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424, and pharmacologic approaches restore CD95-mediated apoptosis in CLL cells and other cancer cells, pointing to an important regulatory role of APTs in CD95 apoptosis. The identification of the depalmitoylation reaction of CD95 by APTs as a microRNA (miRNA) target provides a novel molecular mechanism for how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel posttranslational modification in CLL, which might impact on localization, mobility, and function of molecules, survival signaling, and migration.

CD4+ T cell counts reflect the immunosuppressive state of CD4 helper cells in patients after allogeneic stem cell transplantation.

The recovery of the host immune system after allogeneic hematopoietic stem cell transplantation is pivotal to prevent infections, relapse, and secondary malignancies. In particular, numerical CD4+ T cells reconstitution is delayed and CD4 helper cell function is considered impaired as a consequence of the transplant procedure and concomitant immunosuppressive medication. From HIV/AIDS patients, it is known that numerical and functional CD4 defects increase the risk of opportunistic infections. However, and in contrast to patients with HIV, anti-infective prophylaxis after allogeneic transplantation is usually given for 6 months depending on immunosuppressive medication and existing graft-versus-host disease but independently of absolute CD4+ T cells counts. We hypothesized that a qualitative T cell defect is existing after allogeneic transplantation, especially in patients with delayed immune-reconstitution. Applying transcriptional as well as functional approaches, we show that CD4+ T cells with delayed recovery have a distinct transcriptional profile and cluster differently from T cells originated from patients with completed immune recovery. Moreover, inhibitory signatures are substantially enriched within the transcriptional profile of these T cells translating to functional defects and impaired interleukin 2 production. In addition to time after transplant, CD4+ T cells numbers should be considered for the decision to stop or maintain antimicrobial prophylaxis in patients after allogeneic stem cell transplantation.

Sensitizing protective tumor microenvironments to antibody-mediated therapy.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment refractory B cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF, and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemoimmunotherapeutic regimen represents a potent strategy for using conventional anticancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.