Abstract Interferon‐α (IFNα) remains a potent therapeutic option for myeloproliferative neoplasms (MPNs) with an activated JAK/STAT signaling axis. However, variable patient responses highlight the need for optimized combination strategies. Recent studies suggest a link between cyclin‐dependent kinase 6 (CDK6) and IFN signaling. Here, we investigated whether CDK6 inhibition might play a role in IFN responsiveness in MPN cells. Using CALRdel52 knockin mice, we observed that genetic ablation of Cdk6 resulted in a reduction of spleen weight and platelet counts, while concurrently inducing interferon‐associated transcriptional programs and upregulation of interferon‐alpha receptor 1 (IFNAR1) on MPN cells. CDK6‐deficient CALRdel52 hematopoietic stem and progenitor cells (HSPCs) exhibited increased apoptosis and reduced proliferation upon inflammatory challenge compared to wild‐type CALRdel52 cells, positioning CDK6 as a brake on IFN signaling. Pharmacologic inhibition of CDK6 using palbociclib synergized with pegylated IFNα (pegIFNα), resulting in growth inhibition of MPN cells in vitro and in vivo. In MPN patient samples, lower CDK6 expression was associated with increased IFNAR1 expression and with stronger responses to the palbociclib/pegIFNα combination. Importantly, dose reduction of both palbociclib and pegIFNα maintained efficacy in MPN samples while minimizing cytotoxicity in control hematopoietic cells, revealing a favorable therapeutic window. These findings highlight the potential of combining CDK6 inhibition with pegIFNα to enhance anti‐neoplastic effects in MPNs and support a novel potential approach to improve MPN therapy.

Objective:The expanding field of hematopoietic cell transplantation (HCT) for non-malignant diseases, including those amenable to gene therapy or gene editing, faces challenges due to limited donor availability and the toxicity associated with cell collection methods. Umbilical cord blood (CB) represents a readily accessible source of hematopoietic stem and progenitor cells (HSPCs); however, the cell dose obtainable from a single cord blood unit is frequently insufficient. This limitation can be addressed by enhancing the potency of HSPCs, specifically their capacity to reconstitute hematopoiesis. In our study, we investigated the combined effects of treprostinil, a prostaglandin analog, and cinacalcet, a calcium-sensing receptor modulator, on the reconstitution of hematopoiesis. Methods:A Lineage Cell Depletion Kit was employed to isolate lineage-negative (lin-) HSPCs from mouse bone marrow. A Human CB CD34 Positive Selection Kit was utilized to isolate CD34+ cells from the CB of healthy donors. In vitro, the effects of treprostinil, cinacalcet, and their combination on the migration, adhesion, and differentiation of HSPCs were assessed. In vivo, homing and engraftment were examined. Eight-week-old female and male C57BL/6J, BALB/c, or female NSG mice served as recipient models. Results:When administered concomitantly, treprostinil and cinacalcet exhibited mutual antagonism: the survival of recipient animals was lower when both drugs were administered together compared to either agent alone. Conversely, a sequential regimen involving priming with treprostinil/forskolin followed by cinacalcet treatment in vivo enhanced survival, irrespective of whether hematopoiesis was reconstituted by human or murine HSPCs. In vitro assays demonstrated enhanced migration and adhesion in response to the presence of treprostinil and cinacalcet, suggesting potential synergistic effects. Colony formation confirmed synergism. Conclusion:Augmenting the bone marrow reconstitution potential of HSPCs with treprostinil and cinacalcet shows promise for rescuing patients undergoing HCT. This approach is particularly beneficial for those patients at high risk of transplant failure due to limited numbers of available HSPCs. Furthermore, enhancing the potency of HSPCs has the potential to alleviate the burden and risks associated with HSPC donation, as it would reduce the number of cells needed for collection.

Tyrosine kinase 2 (TYK2) deficiency and loss or inhibition of kinase activity in men and mice leads to similar immune compromised phenotypes, predominantly through impairment of interferon (IFN) and interleukin 12 family responses. Here we relate the transcriptome changes to phenotypical changes observed in TYK2-deficient (Tyk2−/−) and TYK2 kinase-inactive (Tyk2K923E) mice in naïve splenic immune cells and upon ex vivo IFN treatment or in vivo tumor transplant infiltration. The TYK2 activities under homeostatic and both challenged conditions are highly cell-type-specific with respect to quantity and quality of transcriptionally dependent genes. The major impact of loss of TYK2 protein or kinase activity in splenic homeostatic macrophages, NK and CD8+ T cells and tumor-derived cytolytic cells is on IFN responses. While reportedly TYK2 deficiency leads to partial impairment of IFN-I responses, we identified cell-type-specific IFN-I-repressed gene sets completely dependent on TYK2 kinase activity. Reported kinase-inactive functions of TYK2 relate to signaling crosstalk, metabolic functions and cell differentiation or maturation. None of these phenotypes relates to respective enriched gene sets in the TYK2 kinase-inactive cell types. Nonetheless, the scaffolding functions of TYK2 are capable to change transcriptional activities at single gene levels and chromatin accessibility at promoter-distal regions upon cytokine treatment most prominently in CD8+ T cells. The cell-type-specific transcriptomic and epigenetic effects of TYK2 shed new light on the biology of this JAK family member and are relevant for current and future treatment of autoimmune and inflammatory diseases with TYK2 inhibitors.

Background Physiological hematopoiesis relies on a tightly balanced equilibrium of mature blood cell formation and stem cell maintenance that is governed by cell-intrinsic as well as cell-extrinsic stimuli. While the role of transcription factors is well established in this model, the latter are exerted by a complex network of multiple cell types (such as fibroblasts, endothelial cells, bone lining cells, monocytes/macrophages, megakaryocytes, etc.) forming a functional unit that provides the bone marrow (BM) niche. Leukemogenesis is classically perceived as a multi-step process with serial acquisition of mutations at the stem and progenitor cell level ultimately leading to a differentiation block and uncontrolled accumulation of immature cells. However, niche contributions to this process are increasingly accepted. Pronounced alterations of the BM niche were shown under stress and disease conditions, including recent work at the single-cell level. The majority of these studies focused on CD45- stromal niche populations, systematically leaving aside monocytic cells that are also members of the BM niche. We sought to characterize the role of monocytes/macrophages in the BM niche and explore a pathway through which the hematopoietic system potentially reshapes its own niche. Methods We use a novel mouse model of myelodysplastic syndrome (MDS) / chronic myelomonocytic leukemia (CMML) that harbours point mutations in the C/EBP binding sites at the -14kb enhancer element of Pu.1 (subsequently called Pu.1Ki/Ki). The mouse model was validated by ChIP, and extensively characterized using high-throughput multi-omics techniques and a series of transplantation experiments. Results Young Pu.1Ki/Ki mice displayed no overt signs of disease, but a markedly altered monocyte subset homeostasis in favour of the Ly6C+ inflammatory monocyte subset in the BM and peripheral blood (PB). This inflammatory monocyte signature also emerged as a hallmark when the BM plasma was characterized using an unbiased proteomics approach. Phenotypically this translated to an osteoporotic bone phenotype secondary to an increase in osteoclasts, proving BM niche changes on the molecular and microscopic level. Similar results were obtained with a monocyte-specific Pu.1 knockout mouse model. Upon longer follow-up, aged Pu.1Ki/Ki mice developed a myelodysplastic phenotype characterized by thrombopenia, neutropenia and BM hypercellularity resembling human chronic myelomonocytic leukemia (CMML). Of these, thrombopenia typically arose first as an early finding of disease. The CMML phenotype was not associated with accumulation of mutations in typical myeloid candidate genes, making us speculate about a potential role of the niche in disease development. In a series of transplant experiments, myelodysplasia (evidenced by thrombopenia and leukopenia) developed in healthy wildtype (WT) bone marrow, when WT LSK were transplanted into Pu.1Ki/Ki recipients. Importantly, co-transplantation of Pu.1Ki/Ki common monocyte progenitors (cMoP) was required to keep up monocyte-driven niche changes. Conversely, transplantation of Pu.1Ki/Ki LSK started the cascade of disturbed monocyte subset homeostasis, niche changes and increased bone turnover. Likewise, monocyte-induced niche changes fostered proliferation and disease aggressiveness of MLL-AF9 murine leukemia in vitro and in vivo. Importantly, the Pu.1Ki/Ki niche demonstrated an enhanced potential to initiate overt leukemia when the MLL-AF9 transplant was performed in a limiting dilution setup. Summary We delineate a novel disease mechanism that originates from Ly6C+ inflammatory monocytes that are part of the BM niche. In analogy to solid tumors, we refer to these cells as leukemia-associated monocytes (LAM) because of their capability to i) induce myelodysplasia in healthy BM, ii) increase aggressiveness of the oncogene-driven MLL-AF9 leukemia model, and iii) induce leukemia by lowering the leukemia initation potential of the niche.

Background Hematopoietic stem and progenitor cell (HSPC) transplantation (HCT) has significantly advanced over the past decades and is now a standard treatment for various life-threatening conditions. HCT involves harvesting CD34+ HSPCs from sources such as bone marrow (BM), peripheral blood (PB), or umbilical cord blood (CB). However, this process can be physically demanding for stem cell donors, and both PB and BM transplants pose a risk of graft-versus-host disease (GvHD) due to the presence of T cells in the transplant. CB offers advantages with lower risks for donors and a reduced incidence of GvHD, but it is limited by the small number of HSPCs available in a single CB unit. Despite these challenges, HCT remains the most successful somatic cell therapy and, due to its well-established medical use, does not require regulatory approval. This exemption, however, does not apply to primed HSPCs, which must comply with regulatory standards for safety, efficacy, and good manufacturing practices, like traditional small molecules or therapeutic proteins. Repurposing already clinically approved drugs is beneficial as it leverages existing safety and efficacy data, significantly reducing the time and costs required for drug development. In this study, we utilized two approved drugs, treprostinil and cinacalcet, which target distinct signaling pathways in HSPCs, conducting a comprehensive analysis of their single and combined effects. We defined an optimal treatment regimen that enhances the efficacy of all HCTs, regardless of HSPC source, thereby addressing several limitations in current treatments. Methods Lineage-negative (lin-) HSPCs were isolated from murine BM, and CD34+ cells were obtained from the CB of healthy donors. The effects of treprostinil and cinacalcet, both individually and in combination, were assessed by analyzing the migration, adhesion, and differentiation of HSPCs in vitro. In vivo, NSG or NSG-S mice were (xeno)transplanted, and homing and engraftment were evaluated using flow cytometry in limiting cell number conditions. Long-term reconstitution capacity was determined through replating assays and secondary transplantation in animals. Results The experiments provided four major insights: (i) treprostinil was more effective than cinacalcet in stimulating the migration of human HSPCs. (ii) cinacalcet was superior in promoting adhesion. (iii) treprostinil and cinacalcet were mutually antagonistic when used together. (iv) a transient rise in cAMP levels, induced by priming with treprostinil/forskolin, was sufficient to promote the subsequent differentiation of early progenitors (CFU-GEMMs) but impaired the outgrowth of the erythroid lineage (BFU-Es). These insights allowed us to exploit the unique properties of each agent to define an optimal drug regimen for HCT. This regimen involved the sequential priming of murine or human HSPCs with treprostinil and forskolin, followed by 10 days treating recipient animals with cinacalcet. This approach resulted in accelerated recovery of peripheral blood cells and 100% survival of transplanted mice. Moreover, the long-term reconstitution capacity of transplanted HSPCs was elevated. Summary Our findings suggest that the sequential use of treprostinil and cinacalcet as the first “rapid engraftment regimen” can enhance the efficacy of all HCTs, regardless of the HSPC source, and potentially rejuvenate cord blood hematopoietic cell transplantation. This regimen boosts HSPC potency, improves bone marrow reconstitution, and enhances homing and engraftment properties both in vitro and in vivo. It offers two key benefits - i) enabling successful transplants with sub-threshold HSPC collections and ii) reducing the number of cells needed from donors, thereby minimizing the risks and discomfort associated with harvesting. These advantages increase the pool of willing donors and broaden the use of HCT in various clinical settings. Future studies will investigate the safety and mechanisms of this regimen, potentially transforming HCT by making it safer and more accessible for patients and donors.

Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin dependent-kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. We here describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, proposing a concept where MAZ and NFY-A are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes.

The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3ζ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats.

Despite major therapeutic advances in the treatment of acute lymphoblastic leukemia (ALL), resistances and long-term toxicities still pose significant challenges. Cyclins and their associated cyclin-dependent kinases are one focus of cancer research when looking for targeted therapies. We discovered cyclin C to be a key factor for B-cell ALL (B-ALL) development and maintenance. While cyclin C is not essential for normal hematopoiesis, CcncΔ/Δ BCR::ABL1+ B-ALL cells fail to elicit leukemia in mice. RNA sequencing experiments revealed a p53 pathway deregulation in CcncΔ/Δ BCR::ABL1+ cells resulting in the inability of the leukemic cells to adequately respond to stress. A genome-wide CRISPR/Cas9 loss-of-function screen supplemented with additional knock-outs unveiled a dependency of human B-lymphoid cell lines on CCNC. High cyclin C levels in B-cell precursor (BCP) ALL patients were associated with poor event-free survival and increased risk of early disease recurrence after remission. Our findings highlight cyclin C as a potential therapeutic target for B-ALL, particularly to enhance cancer cell sensitivity to stress and chemotherapy.

Quantification of NK cell numbers and their maturation stages from Fig. 1

Supplementary Figure S1. Stat5 expression correlates with the expression of anti-apoptotic genes. Supplementary Figure S2. STAT5-deficient NK cells possess aberrant transcription factors expression. Supplementary Figure S3. Loss of STAT5 alters granzyme, perforin and IFN-gamma production. Supplementary Figure S4. STAT5-deficient NK cells induce tumor promotion. Supplementary Figure S5. Loss of STAT5 increases expression of the pro-angiogenic factor VEGF-A. Supplementary Figure S6. Peripheral splenic NK cells produce VEGF-A which does not impact NK cell numbers, proliferation or maturation but enhances tumor progression in lymphoid tumor models. Supplementary Figure S7. Tumor-infiltrating NK cells and macrophages are localized around blood vessels. Supplementary Figure S8. IL-2 stimulated human NK cells mirror the murine data.

S2. Quantification of perforin and granzyme B western blot from Fig. 2 and ex vivo cytotoxicity assay.

Supplementary Methods, Figure Legends

Quantification of frequency and expression of receptors from Fig. 1F

Background: Cell cycle kinase CDK6 acts as a transcriptional regulator and links cell cycle progression to differentiation and cellular homeostasis. CDK6 has been implicated in the initiation and progression of lymphoid and myeloid malignancies and regulates the balance of long-term hematopoietic stem cell (LT-HSC) dormancy and activation. CDK6 acts in a kinase-dependent and kinase-independent way to regulate haematopoiesis. Aims: As CDK4/6 inhibitors have entered the clinics, we aim at understanding and dissecting kinase-dependent and independent-functions of CDK6 in adult HSCs with application of different stress conditions. Methods: To investigate kinase-independent effects of CDK6 in HSCs, we made use of a mouse model harbouring a kinase inactivated mutant of CDK6 (Cdk6KM/KM). We performed in vivo serial transplantation and homing assays to study HSC re-population capacity. Low-input RNA-Seq of LT-HSCs after the 2nd serial transplantation round was used to understand transcriptional effects. Stressed haematopoiesis was further induced by chemical treatment with polyI:C. Results:Cdk6-/- HSCs are impaired in their ability to re-populate. This was not the case for Cdk6KM/KM hematopoietic cells evidenced by re-plating assays in vitro and serial transplantation settings in vivo. Cdk6KM/KM derived LT-HSCs maintain a stem-cell signature and express genes indicative for HSC dormancy at high levels compared to wild type LT-HSCs. Transcriptional analysis pointed to an altered and improved homing of Cdk6KM/KM LT-HSCs, which was confirmed in homing assays in vivo. This indicates a repressive role of CDK6 in stem cell homing. Upon polyI:C treatment, Cdk6KM/KM mice reacted with decreased stimulation as more Lin-Sca-1+c-kit+ (LSK) cells were residing in the bone marrow. Summary/Conclusion: Our data uncovers kinase-dependent and kinase-independent functions of CDK6 for HSC self-renewal and homing. Whereas serial transplantation settings and homing depend on kinase-inactive CDK6, poly-I:C treatment relies on kinase-activity.

Proceedings of the Annual Meeting of the Austrian Society of Haematology and Medical Oncology Frühjahrstagung 2022 der Österreichischen Gesellschaft für Hämatologie und Medizinische Onkologie und der AHOP – Arbeitsgemeinschaft hämatologischer und onkologischer Pflegepersonen in Österreich Graz, 8.–10. April 2022 Tagungspräsident*In: Univ.-Prof.in Dr.in Hildegard Greinix Univ.-Prof. Dr. Philipp Jost Tagungssekretariat: Assoz.-Prof.in Priv.-Doz.in Dr.in Marija Balic Assoz.-Prof. DDr. Martin Pichler Priv.-Doz. DDr. Eduard Schulz Dr.in Barbara Uhl Klinische Abteilung für Hämatologie / Klinische Abteilung für Onkologie Medizinische Universität Graz LKH-Universitätsklinikum Graz

Background: Background: Cell-cycle progression is governed by regulatory proteins including cyclin-dependent kinases (CDKs), cyclins and CDK inhibitors. The INK4 family comprises p16 INK4a , p15 INK4b , p18 INK4c and p19 INK4d which are CDK4/6 specific inhibitors. Different tumor types display deletion, mutation, or promoter hypermethylation of the INK4 proteins resulting in enhanced CDK4/6 activity. The INK4a-ARF-INK4b locus encodes for p16 INK4a and p15 INK4b and for the tumor suppressor protein p14 ARF (p19 ARF in the mouse) and is one of the most frequently mutated or epigenetically silenced site in human malignancies. CDK4 R24C /CDK6 R31C double knock-in mice, where R24C and R31C mutations make CDK4/6 fully insensitive to INK4 mediated inhibition, show a shortened survival caused by the onset of a variety of tumors including hematopoietic malignancies. This shows the vital importance of a tight controlled inhibition of CDK4/6 activity in hematopoiesis. Aims: Aims: We here explore the consequences of INK4 inhibitory loss on CDK4/6 on the hematopoietic stem cell (HSC) compartment using CDK4 R24C /CDK6 R31C mice. We propose that INK factors regulate important functions in HSCs by direct binding to CDK4/6. This knowledge is required to understand how to best employ CDK inhibitors in therapeutic settings. Methods: Methods: We analyzed the composition of the hematopoietic compartment of CDK4 R24C /CDK6 R31C double knock-in mice under homeostatic conditions. Long-term self-renewal capacity of CDK4 R24C /CDK6 R31C bone marrow (BM) cells was assessed by serial bone marrow transplantation (BMT). was used to investigate the molecular mechanisms underlying the improved self-renewal of CDK4 R24C /CDK6 R31C HSCs. Using the stem/progenitor cell line HPC LSK enabled us to perform ChIP qPCR to understand the involvement of CDK4 R24C /CDK6 R31C for Id1 transcriptional regulation. qHSCs/MPP1 CDK4 R24C /CDK6 R31C this phenotype improved repopulation CDK4 R24C R31C BM INK4 binding to dictates its function in regulating

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal‐regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell‐mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response‐related genes, including interferon‐gamma (IFNγ). We also revealed that NK cells are required for these anti‐metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell‐dependent anti‐tumor immunity and that ASK1‐targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.

Listeria monocytogenes (L. monocytogenes) is a food-borne bacterial pathogen. Innate immunity to L. monocytogenes is profoundly affected by type I interferons (IFN-I). Here we investigated host metabolism in L. monocytogenes-infected mice and its potential control by IFN-I. Accordingly, we used animals lacking either the IFN-I receptor (IFNAR) or IRF9, a subunit of ISGF3, the master regulator of IFN-I-induced genes. Transcriptomes and metabolite profiles showed that L. monocytogenes infection induces metabolic rewiring of the liver. This affects various metabolic pathways including fatty acid (FA) metabolism and oxidative phosphorylation and is partially dependent on IFN-I signaling. Livers and macrophages from Ifnar1-/- mice employ increased glutaminolysis in an IRF9-independent manner, possibly to readjust TCA metabolite levels due to reduced FA oxidation. Moreover, FA oxidation inhibition provides protection from L. monocytogenes infection, explaining part of the protection of Irf9-/- and Ifnar1-/- mice. Our findings define a role of IFN-I in metabolic regulation during L. monocytogenes infection. Metabolic differences between Irf9-/- and Ifnar1-/- mice may underlie the different susceptibility of these mice against lethal infection with L. monocytogenes.