Diffuse large B cell lymphoma (DLBCL) is the most prevalent lymphoid malignancy in adults and exhibits significant heterogeneity. Historically classified into activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes based on cell-of-origin, DLBCL has been further stratified into distinct clusters (A53, ST2, N1, BN2, EZB, and MCD) according to tumor mutational signatures. Although frontline chemo-immunotherapy achieves cure rates of approximately 65%, management of relapsed or refractory disease presents significant challenges, especially in ABC-DLBCL and MCD subtypes. The transcriptional repressor interferon regulatory factor 2 binding protein 2 (IRF2BP2) undergoes frequent mutation in MCD patients, with most mutations anticipated to confer loss-of-function. Consistent with findings in human DLBCL, Irf2bp2 emerges as one of the most commonly co-mutated genes in established MCD mouse models featuring B cell-specific Prdm1 loss, mutant Myd88p.L252P expression, BCL2 overexpression, and Cd79b ITAM mutations. Despite this prevalence, the functional significance of IRF2BP2 in lymphoma pathogenesis and its potential tumor suppressor role in DLBCL remain unexplored. To investigate the landscape of IRF2BP2 DNA binding sites in DLBCL, we performed a series of IRF2BP2 and H3K27Ac ChIP-seq and ATAC-seq experiments in human ABC/MCD-DLBCL cell lines. We found a strong overrepresentation of interferon regulatory factor (IRF) motifs in IRF2BP2 binding peaks with differential signals in the H3K27Ac and ATAC-seq datasets. IRF motifs are often found upstream of cytokine genes that are crucial for regulating inflammation and immune response. Further multiplex cytokine arrays revealed that secretion of several inflammatory cytokines, including IL-1β, were significantly increased in the IRF2BP2 knockout setting. This increase of IL-1β secretion was further validated via ELISA. At the RNA level, differences in IL-1β expression were unchanged in the IRF2BP2-proficient vs. IRF2BP2-deficient setting, suggesting that the differences we see at the protein-level are a result of differential cleavage of pro-IL-1β. To validate this hypothesis, we found that IRF2BP2-deficient cells exhibited enhanced activation of caspase-1, the IL-1 converting enzyme responsible for processing pro-IL-1β into its secreted bioactive form. Additionally, the genetic loci of several regulators of inflammasome activation are bound by IRF2BP2 and change in H3K27Ac after IRF2BP2 knockout, e.g. ZBP1, TET2 and STK24. Furthermore, we observed that CRISPR/Cas9-mediated IRF2BP2 knockout ABC/MCD-DLBCL cells with increasedIL-1β secretion, also displayed enhanced proliferation and elevated NF-κB signaling compared to IRF2BP2-intact cells. This active IL-1β-NF-κB autocrine signaling pathway in IRF2BP2-deficient cells can be therapeutically exploited, and IRF2BP2-deficient cells demonstrated sensitivity to IL-1β inhibition both in vitro and in vivo, while IRF2BP2-proficient cells remained refractory. Moreover, murine lymphoma cell lines derived from MCD mouse models bearing spontaneous Irf2bp2 mutations also displayed anti-IL1β sensitivity in vitro, with sustained responsiveness over extended culture periods. Overall, these results implicate IL-1β as the principal driver of enhanced NF-κB signaling following IRF2BP2 disruption and suggest anti-IL-1β therapy as a promising therapeutic approach for IRF2BP2-mutant patients.

Diffuse large B cell lymphoma (DLBCL) is a highly heterogeneous disease and is the most common lymphoid malignancy in adults (Swerdlow et al., 2016, Alizadeh et al., 2000). It is traditionally divided into two subtypes based on the cell-of-origin: Activated B cell-like (ABC) DLBCL and germinal center B cell-like (GCB) DLBCL (Alizadeh et al., 2000). DLBCL can be further subdivided into clusters A53, ST2, N1, BN2, EZB, and MCD based on the mutational profiles of the tumors (Schmitz et al., 2018, Wright et al., 2020). While cure rates of ~65% are achievable in DLBCL patients with frontline combination chemo-immune therapy (e.g. R-CHOP), treating relapsed or refractory disease remains a challenge, particularly in ABC-DLBCL and MCD patients (Pfreundschuh et al., 2011, Tilly et al., 2015, Schmitz et al., 2018). Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional repressor that is frequently mutated in MCD patients, with many of these mutations predicted to be loss-of-function (Schmitz et al., 2018). Besides human DLBCL tumors, in previously published MCD mouse models, which harbor a B cell-specific loss of Prdm1, expression of mutant Myd88p.L252P, overexpression of BCL2, and in some cases expression of a Cd79b ITAM mutation, Irf2bp2 is also one of the most frequently co-mutated genes (Knittel et al., 2016, Flümann et al., 2023, Flümann et al., 2024). However, the role of IRF2BP2 in lymphoma biology and as a potential tumor suppressor gene in DLBCL has not been investigated. To understand the role of IRF2BP2 in the germinal center reaction, we analyzed the B cell compartment of autochthonous mice which harbor a B cell specific knockout of Irf2bp2 at a premalignant age. We observed a dramatic decrease in germinal center B cells in Irf2bp2 knockout mice compared to controls, as well as a higher relative memory B cell output and reduced class switch recombination. This preliminary dataset suggests that Irf2bp2 may play a role in the dynamics of the germinal center reaction and B cell differentiation. Moreover, we show that CRISPR/Cas9-mediated loss of IRF2BP2 in human ABC-DLBCL cell lines leads to increased proliferation and NF-KB signaling, compared to IRF2BP2-proficient cells. Additionally, we find that IRF2BP2 knockout ABC-DLBCL cells express higher interleukin-1 beta (IL1β) and are sensitive to anti-IL1β inhibition both in vitro and in vivo, while IRF2BP2-proficient cells remain insensitive. Furthermore, murine lymphoma cell lines derived from MCD mouse models, which harbor a spontaneous Irf2bp2 mutation are also sensitive to anti-IL1β inhibition in vitro and retain sensitivity over several weeks. Our findings suggest that IL1β is the primary mediator of increased NF-KB signaling upon IRF2BP2 perturbation and anti-IL1β therapy could be a potential treatment strategy for IRF2BP2-mutant patients.

Diffuse large B -cell lymphoma (DLBCL) is the most common aggressive lymphoma and constitutes a highly heterogenous disease. Recent comprehensive genomic pro filing revealed the identity of numerous molecularly de fined DLBCL subtypes, including a cluster which is characterized by recurrent aberrations in MYD88 , CD79B, and BCL2 , as well as various lesions promoting a block in plasma cell differentiation, including PRDM1 , TBL1XR1, and SPIB . Here, we generated a series of autochthonous mouse models to mimic this DLBCL cluster and speci fically focused on the impact of Cd79b mutations in this setting. We show that canonical Cd79b immunoreceptor tyrosine -based activation motif (ITAM) mutations do not accelerate Myd88 - and BCL2 -driven lymphomagenesis. Cd79b -mutant murine DLBCL were enriched for IgM surface expression, reminiscent of their human counterparts. Moreover, Cd79b -mutant lymphomas displayed a robust formation of cytoplasmic signaling complexes involving MYD88, CD79B, MALT1, and BTK. These complexes were disrupted upon pharmacological BTK inhibition. The BTK inhibitor -mediated disruption of these signaling complexes translated into a selective ibrutinib sensitivity of lymphomas harboring combined Cd79b and Myd88 mutations. Altogether, this in-depth cross -species comparison provides a framework for the development of molecularly targeted therapeutic intervention strategies in DLBCL.

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Targeting the interaction between leukemic cells and the microenvironment is an appealing approach to enhance the therapeutic efficacy in acute myeloid leukemia (AML). AML infiltration induces a significant release of inflammatory cytokines in the human bone marrow niche which accelerates leukemogenesis. As the transmembrane glycoprotein CD38 has been shown to regulate cytokine release, we assessed the anti-leukemic potential of CD38 inhibition in AML. CD38 expression in AML cells proved to depend on microenvironmental cues and could be significantly enforced through addition of tretinoin. In fact, the anti-CD38 antibody daratumumab showed significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, but with modest cell-autonomous cytotoxic activity and independent of CD38 expression level. In line with a predominantly microenvironment-mediated activity of daratumumab in AML, CD38 inhibition significantly induced antibody-dependent phagocytosis and showed interference with AML cell trafficking in vivo in a xenograft transplantation model, but overall lacked robust anti-leukemic effects.

Objective Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. Methods We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing forPIGAmutational analyses. Results We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of thePIGAgene. One patient with relapsed T-PLL exhibited a singlePIGAmutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three differentPIGAmutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealedPIGAwild-type sequence reads in all 16 patients with samples available for testing. Conclusion These data indicate thatPIGAmutations were acquired during or after completion of alemtuzumab treatment.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy characterized by chemotherapy resistance and a median survival of less than 2 years. Here, we investigated the pharmacological effects of the novel highly specific cyclin-dependent kinase 9 (CDK9) inhibitor LDC526 and its clinically used derivate atuveciclib employing primary T-PLL cells in an ex vivo drug sensitivity testing platform. Importantly, all T-PLL samples were sensitive to CDK9 inhibition at submicromolar concentrations, while conventional cytotoxic drugs were found to be largely ineffective. At the cellular level LDC526 inhibited the phosphorylation at serine 2 of the RNA polymerase II C-terminal domain resulting in decreased de novo RNA transcription. LDC526 induced apoptotic leukemic cell death through down-regulating MYC and MCL1 both at the mRNA and protein level. Microarray-based transcriptomic profiling revealed that genes down-modulated in response to CDK9 inhibition were enriched for MYC and JAK-STAT targets. By contrast, CDK9 inhibition increased the expression of the tumor suppressor FBXW7, which may contribute to decreased MYC and MCL1 protein levels. Finally, the combination of atuvecliclib and the BCL2 inhibitor venetoclax exhibited synergistic anti-leukemic activity, providing the rationale for a novel targeted-agent-based treatment of T-PLL.

In order to accomplish their physiological functions leukocytes have the capability to migrate. As a prerequisite they need to adopt a polarized cell shape, forming a leading edge at the front and a uropod at rear pole. In this study we explored the capability of chronic lymphocytic leukaemia (CLL) cells to adopt this leukocyte-specific migration phenotype. Furthermore, we studied the impact of the Toll-like receptor 9 (TLR9) agonists CpGs type A, B and C and the antagonist oligodesoxynucleotide (ODN) INH-18 on the cell polarization and migration process of primary human CLL cells. Upon cultivation, a portion of purified CLL cells adopted polarized cell shapes spontaneously (range 10-38%). Stimulation with CpG ODNs type B (ODN 2006) and CpGs type C (ODN 2395) significantly increased the frequency of morphologically polarized CLL cells, while ODN INH-18 was hardly able to act antagonistically. Like in human hematopoietic stem and progenitor cells, in morphologically polarized CLL cells CXCR4 was redistributed to the leading edge and CD50 to the uropod. Coupled to the increased frequencies of morphologically polarized cells, CpGs type B and C stimulated CLL cells showed higher migration activities in vitro and following intravenous injection higher homing frequencies to the bone marrow of immunocompromised NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice. Thus, presumably independent of TLR-9 signaling, CpGs type B and C promote the cellular polarization process of CLL cells and their ability to migrate in vitro and in vivo.

Acute myeloid leukemia (AML) is characterized by a high relapse rate and dismal long-term overall survival which is related to persistence of leukemia-initiating cells in their niche. Different animal models of myeloid malignancies reveal how neoplastic cells alter the structural and functional characteristics of the hematopoietic stem cell niche to reinforce malignancy. Understanding and disruption of the microenvironmental interactions with AML cells are a vital need. Malignant niches frequently go along with inflammatory responses, but their impact on cancerogenesis often remains unexplored. Here, we uncovered an aberrant production of inflammatory cytokines in untreated AML bone marrow that was proved to promote the proliferation of leukemia cells. This inflammatory response induced an activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in AML blasts as well as bone marrow stromal cells that also fostered leukemia proliferation. Inhibition of JAK/STAT signaling using the selective JAK1/2 inhibitor ruxolitinib resulted in significant antileukemic activity in AML in vitro which is mediated through both cell-autonomous and microenvironment-mediated mechanisms. However, in a xenograft transplantation model, monotherapy with ruxolitinib did not achieve substantial antileukemic activity, possibly suggesting a complementary function of JAK1/2 inhibition in AML.

Endothelial and mesenchymal stromal cells (ECs/MSCs) are crucial components of hematopoietic bone marrow stem cell niches. Both cell types appear to be required to support the maintenance and expansion of multipotent hematopoietic cells, i.e. hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). With the aim to exploit niche cell properties for experimental and potential clinical applications, we analyzed the potential of primary ECs alone and in combination with MSCs to support the ex vivo expansion/maintenance of human hematopoietic stem and progenitor cells (HSPCs). Even though a massive expansion of total CD34+ HSPCs was observed, none of the tested culture conditions supported the expansion or maintenance of multipotent HSPCs. Instead, mainly lympho-myeloid primed progenitors (LMPPs) were expanded. Similarly, following transplantation into immunocompromised mice the percentage of multipotent HSPCs within the engrafted HSPC population was significantly decreased compared to the original graft. Consistent with the in vitro findings, a bias towards lympho-myeloid lineage potentials was observed. In our conditions, neither classical co-cultures of HSPCs with primary ECs or MSCs, even in combination, nor the xenograft environment in immunocompromised mice efficiently support the expansion of multipotent HSPCs. Instead, enhanced expansion and a consistent bias towards lympho-myeloid committed LMPPs were observed.

Introduction: The thymus is a specialized hematopoietic organ, which is responsible for the generation of T cells. The central thymic cell type controlling T cell development are thymic epithelial cells (TECs). Based on their specific function and anatomic location TECs are separated into cortical and medullary subsets (cTECs and mTECs). cTECs express pivotal NOTCH-ligands such as DLL4 controlling T cell lineage commitment while mTECs play a central role in negative selection of developing T cells. Acquisition of NOTCH1 gain-of-function mutations play a central role in acute T cell lymphoblastic leukemia (T-ALL) development. During T-ALL leukemogenesis aberrant expression of transcription factors such as SCL/TAL1 and LMO1 block T cell differentiation and increase self-renewal while NOTCH1 mutations promote survival and proliferation. Since most acquired NOTCH1 mutations still require ligand binding to exert augmented signaling we propose DLL4-expressing TECs playing a critical role during T-ALL leukemogenesis. Methods: In the present study, we used a Scl/Lmo1 T-ALL transgenic mouse model, murine ANV and TE71 TEC cell lines and human T-ALL cell lines (Jurkat, ALL-SIL, DND-41, and HPB-ALL) to investigate TEC dynamics and function in the T-ALL context. Results: First, we demonstrated T-ALL supporting potential of TEC cell lines in vitro, which was comparable to the mesenchymal cell line OP9. Next, we showed in the Scl/Lmo1 T-ALL mouse model which had a mean survival rate of 90 days that preleukemic thymocytes displayed a striking upregulation of Notch1 target genes. Interestingly, fluorescence microscopy revealed a relative expansion of cortical and a relative reduction of the medullary thymic areas in Scl/Lmo1 thymi (Fig. 1A). Correspondingly, absolute numbers of cTECs expanded while mTEC numbers declined (Fig. 1B). Gene expression profiling of sorted preleukemic Scl/Lmo1 cTECs revealed upregulation of the chemokine CXCL10 (Fig. 1C). Moreover, increased CXCL10 chemokine concentrations were detected in Scl/Lmo1 thymic interstitial fluid (Fig.1D). Strikingly, we demonstrated T-ALL dependence of TEC Cxcl10 upregulation. We showed that Cxcl10 upregulation in TEC cell lines was only induced by direct cellular contact with Scl/Lmo1 thymocytes while wild-type control thymocytes did not alter TEC cell line Cxcl10 expression (Fig. 1E). Next, a high proportion of the CXCL10 receptor CXCR3 expressing cells was revealed in Scl/Lmo1 thymi (Fig. 1F) and by human T-ALL cell lines. Finally, we demonstrated a CXCL10 dependent pro-survival effect within cultured SCL/LMO1 thymocytes (Fig. 1G), which was associated with the activation of NOTCH1 signaling (Fig. 1H). Conclusions: In summary, the data support a novel T-ALL-promoting regulatory circuit in which emerging T-ALL lymphoblasts induce CXCL10 in expanding TECs which positively feeds back to T-ALL cells via the CXCL10 receptor CXCR3. Disclosures Dührsen: Celgene: Research Funding; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Roche: Honoraria, Research Funding; CPT: Consultancy, Honoraria; Janssen: Honoraria. Göthert:Proteros Biostructures: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel support; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Other: Travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel support; AOP Orphan Pharmaceuticals: Honoraria, Other: Travel support.

Onset of progression even during therapy with novel drugs remains an issue in chronic lymphocytic leukemia (CLL). Thus, there is ongoing demand for novel agents. Approaches targeting cyclin-dependent kinases (CDK) have reached the clinical trial stage. CDK9 mediating RNA transcriptional elongation is the evolving pivotal CLL CDK inhibitor target. However, more CDK9 selective compounds are desirable. Here, we describe the CDK9 inhibitor LDC526 displaying a low nanomolar biochemical activity against CDK9 and an at least 50-fold selectivity against other CDKs. After demonstrating in vitro MEC-1 cell line and primary human CLL cell cytotoxicity we evaluated the LDC526 in vivo effect on human CLL cells transplanted into NOD/scid/γcnull (NSG) mice. LDC526 administration (75 mg/kg) for 5 days resulted in a 77% reduction of human CLL cells in NSG spleens compared to carrier control treatment. Next, we longitudinally studied the LDC526 impact on circulating CLL cells in the TCL1 transgenic mouse model. LDC526 (50 mg/kg) administration for two days led to a 16-fold reduction of blood CLL cell numbers. Remarkably, residual CLL cells exhibited significantly increased intracellular BCL-2 levels. However, the LDC526 cytotoxic effect was not restricted to CLL cells as also declining numbers of normal B and T lymphocytes were observed in LDC526 treated TCL1 mice. Taken together, our in vivo data provide a strong rational for continued LDC526 development in CLL therapy and argue for the combination with BCL-2 inhibitors.

The Wnt signalling pathway, one of the core de-regulated pathways in chronic lymphocytic leukaemia (CLL), is activated in only a subset of patients through somatic mutations. Here we describe alternative, microenvironment-dependent mechanisms of Wnt activation in malignant B cells. We show that tumour cells specifically induce Notch2 activity in mesenchymal stromal cells (MSCs) required for the transcription of the complement factor C1q. MSC-derived C1q in turn inhibits Gsk3-β mediated degradation of β-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises β-catenin. Together, these stroma Notch2-dependent mechanisms induce strong activation of canonical Wnt signalling in CLL cells. Pharmacological inhibition of the Wnt pathway impairs microenvironment-mediated survival of tumour cells. Similarly, inhibition of Notch signalling diminishes survival of stroma-protected CLL cells in vitro and disease engraftment in vivo. Notch2 activation in the microenvironment is a pre-requisite for the activation of canonical Wnt signalling in tumour cells.

Introduction: Epigenetic regulatory mechanisms such as the modification of histone residues are perturbed in myeloid neoplasms. Amongst others, the methylation status of Lysine residue 4 on Histone 3 (H3K4) represents one of the central dysregulated histone marks in acute myeloid leukemia (AML). Demethylation of H3K4 is mediated by the Lysine (K)-specific demethylase 1A (LSD1/ KDM1A). Remarkably, LSD1 expression is de-regulated in AML and therefore represents an attractive therapeutic target. In fact, pharmacologic LSD1 inhibition was shown to induce AML cell differentiation and apoptosis in combination with all-trans-retinoic acid (ATRA) exposure. Recently, a phase 1 trial with the irreversible LSD1 inhibitor IMG-7289 (IMG) ± ATRA in patients with advanced myeloid malignancies commenced (ClinicalTrials.gov Identifier: NCT02842827). However, the effect of a LSD1-inhibitory therapy on normal human progenitors has not been studied in detail. Methods: Experiments were performed with mobilized CD34+ cells of deceased multiple myeloma and lymphoma patients. The usage of these cells was approved by the local ethics committee. The following experimental approaches were employed: (1) studying in vitro colony forming unit (CFU) potential of human CD34+ progenitors in the presence of increasing IMG concentrations, (2) investigating the CFU potential in re-plating experiments of primary IMG-7289 exposed CD34+ cell CFU cultures and (3) studying the impact of IMG on in vitro cytokine (SCF, IL-3, IL-6, Flt3L and TPO for 7 days)-driven CD34+ progenitor expansion. Results: Performing CFU assays in the presence of increasing IMG concentrations revealed a shift from diverse colony types in DMSO control treated CFU assays to increasing phenotypic monocytic (CFU-M) and decreasing granulocytic (CFU-G) and erythroid (BFU-E) colony types (Fig. 1A). Accordingly, increased CFU-M and decreased CFU-G and BFU-E colony numbers were observed (Fig. 1B). In contrast, sole ATRA treatment abrogated CFU-M colony formation, increased CFU-G and slightly decreased BFU-E colony numbers (Fig. 1B). To study the impact of LSD1 inhibition in synergy with ATRA-driven differentiation therapy we treated CFU assays with increasing IMG concentrations in combination with a constant ATRA concentration (10 nM). Strikingly, IMG and ATRA co-exposure severely depleted CFU-M and BFU-E colonies while preserving CFU-G colony formation (Fig.1B). In order to study the impact of IMG on progenitor self-renewal we re-plated IMG-exposed CFU assays in the absence of IMG. In the re-plated dishes we observed a 2.9- and 3.7-fold increase of CFU-G colony numbers compared to the primary CFU-G IMG exposed (100 nM and 1µM, respectively) colony numbers. Finally, we investigated the impact of IMG exposure on in vitro cytokine-driven CD34+ progenitor expansion. In DMSO treated cultures 7 day cytokine incubation led to a 37-fold expansion of CD34+ cell numbers while increasing the total CFU numbers 1.8-fold. Compared to the DMSO control cultures, IMG exposure (100nM and 1µM) boosted CFU-M colony numbers 1.7- and 2.3-fold, respectively. Strikingly, also CFU-G colony numbers were slightly increased by IMG exposure (1.3- and 1.6-fold at 100nM and 1µM, respectively). To investigate how IMG exposure during cytokine-driven CD34+ progenitor expansion alters the transcriptional program of human CD34+ progenitors we analyzed the expression of the hematopoietic regulatory genes GFI1, GFI1B, IRF8 and RARA by real-time PCR. IMG exposure (100 nM) increased the expression of these genes 2.2-, 1.7-, 1.9- and 1.6-fold, respectively. De-repression of GFI1 and GFI1B expression by LSD1 inhibition was expected and the observed upregulation of the monocytic regulator IRF8 is in accordance with the observed increased monocytic colony numbers under LSD1 inhibition. Lastly, RARA upregulation suggested that LSD1 inhibition promotes retinoic acid receptor signaling. Conclusions: Our findings reveal that pharmacologic LSD1 inhibition increases human monocytic progenitor numbers. Furthermore, in vitro CFU-re-plating and also progenitor expansion experiments point to LSD1 inhibition not depleting granulocytic progenitors. Collectively, our data suggest that expected side effects of a LSD1-targeted therapy such as neutropenia will be reversible. Rienhoff: Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dührsen:Janssen: Honoraria; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Research Funding; Gilead: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Göthert:AOP Orphan Pharmaceuticals: Other: travel support; Proteros Biostructures: Consultancy; Novartis: Honoraria; Pfizer: Consultancy; Incyte: Consultancy, Honoraria, Other: travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: travel support.

Acute myeloid leukemia (AML) is characterized by an early failure of healthy hematopoiesis and a high relapse rate, caused by persistent leukemia initiating cells in the bone marrow niche. The hematopoietic stem cell niche in myeloid malignancies shows severe structural and functional alterations with an inflammatory response upon leukemia infiltration and impaired immunosurveillance. CD38 is a glycoprotein that is expressed on various immunoregulatory cells and commonly on AML blasts. Targeting CD38 by using the monoclonal antibody daratumumab showed high efficacy in multiple myeloma which is mediated by both cell-autonomous as well as immunomodulatory mechanisms. In this study we have investigated the anti-leukemic efficacy of daratumumab as well as its underlying mechanisms in AML.

With great interest, we read the report by Byrd et al. [1] in this journal about lucatumumab (HCD122, formerly CHIR-12.12) in patients with relapsed chronic lymphocytic leukemia (CLL). This phase-I trial [1] concluded an acceptable tolerability and a limited single-agent activity of this CD40 monoclonal antibody (mAb). Two subsequent early-phase studies in relapsed/refractory (r/r) lymphoma [2] and multiple myeloma (MM) [3] confirmed selective response inductions as well as the encouraging safety profile and pharmacodynamics of this agent. We noted the interesting clinical response patterns of nodal debulking versus unaffected peripheral blood (PB) burden reported in CLL and specific lymphoma subsets, i.e., follicular lymphoma (FL). It prompted us to communicate here our novel preclinical observations in CLL and to review reported data on HCD122. Postulating a particular milieu-dictated activity and potential synergies in combined applications of HCD122, we addressed here the effects of lucatumumab at the regenerative niche of CLL and in drug combinations. In a first set of experiments, we employed our established NOD/SCID CLL-xenograft model.[4] The human disease engrafts in spleen without PB lymphocytosis and only when cotransplanted with T-/NK-cells and monocytes. This allows selective therapeutic and analytic access to CLL at its sites of active crosstalk with the CD40L-providing micromilieu while preserving prerequisites for antibody-dependent cellular cytotoxicity (ADCC). Freshly isolated PBMC from four patients with CLL were incubated ex vivo for 15 min with HCD122 (1 mg/kg and 10 mg/kg) or controls (PBS or hu-IgG1 isotype) followed by intravenous injection (100 10 cells) into 6-week-old female hosts (3 animals/patient). After 24 h, we observed a significant (paired t-tests) reduction of human leukocytes (CD45 flow cytometry) and CLL cells (CD5/19 gate) in the splenic suspensions at both HCD122 doses (CD45: HCD122 1 mg/kg: 0.29 ± 0.10, p 1⁄4 0.03; 10 mg/kg: 0.30 ± 0.19, p 1⁄4 0.03 (not shown); CLL cells: HCD122 1 mg/kg: 0.28 ± 0.07, p 1⁄4 0.02; 10 mg/kg: 0.28 ± 0.14, p 1⁄4 0.02 [Figure 1A]; all means ± SEMs). No reductions in splenic T-cell counts or (normal level) PB leukocytes (not shown) were observed. In a modification of this system, HCD122 treatment (days +14 and +21) was commenced after CLL cells had engrafted. Analysis of splenic infiltration at day +28 showed a significant depletion of human CLL cells in both HCD122 dosage groups (1 mg/kg: 0.72 ± 0.34, p 1⁄4 0.04; 10 mg/kg: 0.44 ± 0.19, p 1⁄4 0.03; means ± SEMs [Figure1B]). Although mainly attributable to leukemia cell reduction, the accompanying drop in infiltrating pan-leukocytes (CD45) was paralleled by decreases in infiltrating CD3 T-cells as well (not shown). A series of immunohistochemistries (IHC) on paraffin-embedded portions of these spleens confirmed the results semiquantitatively. The perivascular CLL infiltrates tended to be smaller in the HCD122-groups (as per H&E, CD45, CD19 [Figure 1C], and TCL1 stains), which was associated with slightly less-intense nuclear signals for NFkB-p65. There was no difference in CD38 (flow cytometry) as well as Ki67 and bcl2 signals (IHC). In another modification of the latter xenograft set-up in two additional patients (6 mice), the onset of splenic CLL cell depletion was as early as 2-h post-injection with peaks between 6–24 h. The next set of in vitro experiments on freshly isolated CLL patient cells showed that HCD122 reverses the CD40L-induced resistance to fludarabine cytotoxicity. An

Hematopoietic stem and progenitor cells (HSPCs) can self-renew and create committed progenitors, a process supposed to involve asymmetric cell divisions (ACDs). Previously, we had linked the kinetics of CD133 expression with ACDs but failed to detect asymmetric segregation of classical CD133 epitopes on fixed, mitotic HSPCs. Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs. After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level. Approximately 70% of the HSPCs of the multipotent progenitor (MPP) fraction studied performed ACDs, and about 25% generated lymphoid-primed multipotent progenitor (LMPP) as wells as erythromyeloid progenitor (EMP) daughter cells. Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.

The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However, this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g., LMPPs), implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here, we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP), whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model.

Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin (GRN), is significantly higher expressed in aggressive CD38(+)ZAP-70(+) as compared to indolent CD38(-)ZAP-70(-) chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics (11q-, 17p-) as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn (HR = 2.06, 95%-CI = 1.13-3.76, p = 0.018), unmutated IGHV status (HR = 5.63, 95%-CI = 3.05-10.38, p<0.001), high risk as defined by the study protocol (HR = 2.06, 95%-CI = 1.09-3.89, p = 0.026) but not poor risk cytogenetics were independent prognostic markers. In summary our results suggest that Pgrn is a novel, robust and independent prognostic marker in CLL that can be easily measured by ELISA.