Expansion Of Myeloid Cells Research Articles

Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis

Massive expansion of immature and suppressive myeloid cells is a common feature of malignant solid tumors. Over-expression of cyclin-dependent kinase 20, also known as cell cycle-related kinase (CCRK), in hepatocellular carcinoma (HCC) correlates with reduced patient survival and low immunotherapy responsiveness. Beyond tumor-intrinsic oncogenicity, here we demonstrated that CCRK is upregulated in myeloid cells in tumor-bearing mice and in patients with HCC. Intratumoral injection of Ccrk-knockdown myeloid-derived suppressor cells (MDSCs) increased tumor-infiltrating CD8+T cells and suppressed HCC tumorigenicity. Using an indel mutant transgenic model, we showed that Ccrk inactivation in myeloid cells conferred a mature phenotype with elevated IL-12 production, driving Th1 responses and CD8+T cell cytotoxicity to reduce orthotopic tumor growth and prolong survival. Mechanistically, CCRK activates STAT3/E4BP4 signaling in MDSCs to acquire immunosuppressive activity through transcriptional IL-10 induction and IL-12 suppression. Taken together, our findings unravel mechanistic insights into MDSC-mediated immunosuppression and offer a therapeutic kinase-target for cancer immunotherapy.

Targeting miR-223 enhances myeloid-derived suppressor cell suppressive activities in multiple sclerosis patients.

Multiple sclerosis (MS) is an incurable autoimmune inflammatory demyelinating disease of the central nervous system. Several MS medications can modify disease course through effects on adaptive immune cells, while drugs targeting innate immunity are under investigation. Myeloid-derived suppressor cells (MDSCs) which arise during chronic inflammation, are defined by their T-cell immunosuppressive functions. MiR-223 modulates myeloid cell maturation and expansion, including MDSCs. MDSCs isolated from healthy controls (HC) and people with MS (pwMS) were co-cultured with CD4+ T-cells to study their immunosuppressive activities in vitro. Cytokines and chemokines concentration were evaluated by Luminex assay in the serum of HC, pwMS, and other neuroinflammatory diseases and correlated with MDSC activities. MDSC suppressive functions are dysregulated in pwMS compared to HC, which was reversed by glucocorticoids (GC). GC specifically downregulated miR-223 levels in MDSCs and increased the expression of STAT3. In vitro assay showed that miR-223 inhibition enhanced MDSC suppressive activity, STAT3 dependently. By multiple linear regression analysis, we demonstrated that MDSC phosphorylated STAT3 was correlated with serum GM-CSF in HC and pwMS. These results suggest that miR-223 could be a therapeutic target for enhancing MDSC's suppressive activities as an alternative to GC.

In Silico Post Translational Analysis of Functional Single Nucleotide Alterations in Human TERT Gene Associated with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) refers to a diverse assemblage of hematological malignancies that constitute clonal expansion of immature myeloid progenitor cells in the peripheral blood and bone marrow. TERT gene ensures telomeres maintenance, chromosome stability and prevention of malignancy. The TERT gene has several single nucleotide polymorphisms (SNPs) that have been linked to a number of diseases, including AML. Objective: To classify the harmful TERT gene mutations, and to analyze them using various computational approaches at structural, functional and translational expression levels Methods: National Centre for Biotechnology Information (NCBI) database was used to retrieve nsSNPs of TERT gene (Q53H, V170M, A184T, S255Y, A288V, H412Y, I540M, R631W) reported in AML and they were analyzed using various bioinformatics tools. Results: In this in silico analysis, it was observed that seven out of eight SNPs had a damaging effect; they could affect the protein stability, protein-protein interactions, hydrophobicity, protein folding, three-dimensional structure, secondary structure and conservation profile. 3D models were generated and validated by various tools and the structural effect of these alterations was observed on protein function that was destabilizing to the RNA folding, protein-protein interactions and other functionally associated proteins. Analysis of post translational modifications showed no significant effect of these mutations. Conclusions: These SNPs could be used in future as potential targets in disease diagnosis, biological markers and protein studies.

Abstract A06: Murine models of mutant p53 clonal hematopoiesis elucidate mechanisms of expansion of p53 mutant subclones by MDM2 inhibition

Abstract Unleashing the pro-apoptotic effects of p53 by pharmacologic inhibition of MDM2 has been an attractive therapeutic strategy in hematologic malignancies with intact p53. However, concerns were expressed regarding the selection of TP53-mutant subclones with potential GOF activities. It is not clear whether pre-existing sub-clones with TP53 mutation(s) undergo malignant transformation or clonal expansion after exposure to MDM2i therapy. We generated traceable conditional somatic TP53R172H mutant mouse models to monitor and track the expansion of p53 mutant myeloid cells over the course of therapy with MDM2i. We combined a myeloid-specific Cre line, LysM-CreERT2, or Mx1-cre with a conditional knock-in mutant Trp53 allele that constitutively expresses WT Trp53 and converts to mutant (Mut) Trp53R172H upon Cre-mediated recombination. To trace the recombined hematopoietic cells, we used the Rosa26mTmG reporter allele as previously described and performed a pulse-chase experiment. LysM-CreERT2;mTmG;Trp53R172H/fx mice did not develop leukemia after 1 year upon multiple tamoxifen inductions, suggesting that Trp53 mutation in mature myeloid cells is not leukemogenic. Having a GFP marker in myeloid cells allowed us to monitor the population of GFP+ myeloid cells in peripheral blood after each tamoxifen pulse. The GFP+ cells slowly disappeared from peripheral blood over time and were completely undetectable after 1 month following induction, suggesting that p53 mutation in myeloid cells does not extend the half-life of myeloid cells. Next, to assess if the Mdm2i therapy may cooperate with Trp53 mutation and transform the myeloid cells to AML, we induced the p53 mutation by tamoxifen injection and treated LysM-CreERT2;mTmG;Trp53fx/fx and LysM-CreERT2;mTmG;Trp53R172H/fx mice with the MDM2i, idasanutlin, for 10 days. There were no significant differences in the number of GFP+ myeloid cells between the two groups at baseline, however, in the p53-mutant group, the population of GFP+ cells was significantly decreased and in some mice was fully eradicated after MDM2i treatment. We transplanted LysM-CreERT2;mTmG;Trp53R172H/fx and control mice with syngeneic MLL-AF9 transformed leukemia cells and treated them with idasanutlin 1 week after transplant. Strikingly, the population of GFP+ cells was highly expanded in p53 mutant mice transplanted with AML after MDM2i treatment, suggesting that MDM2 inhibition promotes the proliferation of p53-mutant myeloid subclones in the context of AML. We sorted the GFP+ cells and transplanted them into syngeneic mice. No leukemia arose after transplantation. We here established the first in vivo model of p53-mutant clonal hematopoiesis using a new p53 mutant conditional allele. The expanded population of p53 mutant subclone did not transform into AML. However, in the presence of fully transformed AML cells, the p53 mutant clonal hematopoiesis expanded. This finding is consistent with reports of transitory expansion of p53 mutant cells in patients with myelofibrosis treated with MDM2 inhibitor. Citation Format: Russell Pourebrahim, Rafel Heinz Montoya, Lauren Ostermann, Michael Andreeff. Murine models of mutant p53 clonal hematopoiesis elucidate mechanisms of expansion of p53 mutant subclones by MDM2 inhibition [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A06.

Abstract 3517: Multi-modal single-cell and spatial genomics reveals genomic, adaptive and microenvironmental features of human non-small cell lung cancer brain metastasis

Abstract Non-small cell lung cancer (NSCLC) accounts for nearly half of all newly diagnosed patients with brain metastasis (BM), followed by melanoma and breast carcinomas. The presence of BM is associated with reduced response to several modern cancer therapies and a poor prognosis, but the underlying molecular underpinnings remain poorly understood. Here, we performed multi-modal single-nucleus RNA, T cell receptor, single-cell spatial, and whole-genome sequencing (WGS) of 44 primary NSCLC tumors (PTs) and BMs. Through combination of WGS with inferred copy-number alterations (CNAs) and gene expression snRNA-seq, we robustly identify malignant cells despite the presence of healthy cell mosaicism. We find a strong association of chromosomal instability (CIN) and brain-metastatic organotropism. Through integration with clinical information and thousands of publicly available whole-exome sequencing (WES) profiles obtained from patients with NSCLC, we validate this observation and show that CIN progressively increases from PTs to extracranial metastases (ECMs) and is the highest in BMs. Using non-negative matrix factorization, we identify recurrent transcriptional hallmarks cancer metastasis, and additionally find that cancer cells from BMs strongly enrich for a neuronal-like cell state. At single-cell resolution, we indeed identify a rare cancer cell population genomically define by very high CIN, and transcriptionally characterized by a program of epithelial-to-mesenchymal transition (EMT), neuronal-like differentiation, and loss of lineage attribution. We show in our data and external scRNA-seq data that this cell state does not ecist in healthy lungs, progressively enriches from PTs to ECMs, and is most abundant in BMs, suggesting that these cells may indeed give rise to BMs. Furthermore, through integration of snRNA/TCR-seq and spatial transcriptomics, we find distinct tumor-microenvironments across disease sites, including, nearly exclusive expansion of tissue-resident myeloid cells in PTs, while BMs are largely dominated by dense infiltration with monocyte-derived macrophages and granulocytes, impaired T cell infiltration, activation and clonal expansion. Lastly, spatial transcriptomics also recurrent, cell-type specific patterns of geographic variability in key pathways, including antigen presentation, EMT, oxidative phosphorylation, and inflammatory response and associated cellular micro-niches. Together, this work identifies cellular, genomic, and transcriptional features of NSCLC BMs and has important therapeutic implications for novel therapies, in particular immunomodulatory approaches targeting cell types/states unique to disease sites. Citation Format: Somnath Tagore, Lindsay Caprio, Amit Dipak Amin, Irving Barrera, Johannes Melms, Karan Luthria, Yiping Wang, Yohanna Georgis, Abhi Jaiswal, Galina G. Lagos, Zachary Walsh, Parin Shah, Jana Biermann, Neha Sheikh, Priyanka Ramaradj, Niroshana Anandasabapathy, Hanina Hibshoosh, Gary Schwartz, Brian Henick, Alison Taylor, Fei Chen, Benjamin Izar. Multi-modal single-cell and spatial genomics reveals genomic, adaptive and microenvironmental features of human non-small cell lung cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3517.

Abstract 1101: Combination therapy to overcome radiotherapy induced immunosuppression and improve tumor control

Abstract Background: The relationship between radiation therapy, immunosuppressive myeloid populations and adenosine is not well characterized. Here we test the hypothesis that radiation-induced adenosine signaling promotes tumor growth and causes suppression of T cell function and that therapeutic targeting may improve response to radiotherapy. Methods: Orthotopic 4T1 cells were implanted in Balb/c mice and tumors were irradiated using small animal radiation research platform (SARRP, Xstrahl; 220 kV) with 3 fractions of 8 Gy and/or treated with adenosine receptor 2A and 2B (A2AR/A2BRi) inhibitor, CD73 inhibitor (CD73i), and/or checkpoint inhibitor, aPD-1. Tumor response was assessed by tumor volume and the immune response characterized by flow cytometry. Results: Intratumoral immune response was evaluated 72 hours post-irradiation. Radiotherapy induced an expansion of myeloid cells particularly monocytic cells (13.5% vs 7% of CD45+ cells) with a corresponding contraction of lymphocytes. Monocytes from irradiated tumors had increased expression of most of the key members of the adenosine signaling pathway including enpp1 (2.5% vs 0.6%), CD73 (10.3% vs 4.8%), CD39 (10.7% vs 4.4%), A2aR (2.8% vs 0.6%) and A2bR (2.4% vs 0.3%), compared to untreated controls. In the absence of tumor irradiation, the combination of A2AR/A2BRi & CD73i with αPD-1 showed no improvement in tumor control compared to A2AR/A2BRi & CD73i alone or untreated controls. However, in the setting of tumor irradiation, we measured a significant reduction in 4T1 tumor volume when aPD-1 was added to A2AR/A2BRi & CD73i, compared to A2AR/A2BRi & CD73i alone (156mm^3 vs 250mm^3). The addition of αPD-1 to A2AR/A2BRi & CD73i and tumor irradiation resulted in decrease in monocytic cells (9.4% vs 15% of CD45+ cells) and expression of CD39 (7.4% vs 11%), CD73 (7.5% vs 11%), A2aR (2.2% vs 7%) and A2bR (2.7% vs 9%) in monocytic compartment, compared to radiation and A2AR/A2BRi & CD73i. Further, there was a corresponding expansion of Ki67+ proliferating (52% vs 36%) and IFN-γ producing CD8+ T cells (3.3% vs 2%) after treatment with radiation, A2AR/A2BRi & CD73i, and αPD-1, suggesting improved T cell effector function. Conclusion: Radiation-induced adenosine signaling facilitates immunosuppressive changes in the TME. In the immunotherapy insensitive 4T1 mouse model, adenosine blockade induces checkpoint inhibition sensitivity through alleviation of immunosuppression, thus improving tumor control. Citation Format: Shruti Bansal, Matthew Chaimowitz, Julia Ann, Patrick McCann, Jason Piersaint, Claudia Aiello, Namita Sen, Julia Kogan, Catherine Spina. Combination therapy to overcome radiotherapy induced immunosuppression and improve tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1101.

Abstract 1798: ALDH inhibition as modulator of ovarian tumor associated immune cells

Abstract Rationale: Ovarian cancer (OC) is an aggressive disease with the 3rd highest mortality to incidence ratio of all cancers. Aldehyde dehydrogenase-1A enzymes play a key role in retinoic acid (RA) synthesis. RA regulates cell proliferation, differentiation, and survival by activating nuclear receptor transcription factors (RAR, RXR). In cancer cells, increased ALDH expression correlates with chemoresistance and poor prognosis. Our goal is to effectively utilize ALDH inhibitors (ALDHi) as novel therapeutics and identify their mechanism in cancer and immune cells. Based on RNAseq data, NR4A1 has emerged as a possible ALDHi target. After binding RXRγ, NR4A1 serves as a ‘molecular switch’ for cancer cell survival. Here, we explore the potential use of ALDHi as immune modulators in OC. Methods: T cells: The effect of several ALDHi on T cells following anti-CD3/CD28 polyclonal stimulation was tested via flow cytometry. Macrophages: Human OC ascites samples were cultured and treated for 72h with ALDHi or DMSO control. Flow cytometry was performed to identify ascites resident cell populations. Western blot to detect NR4A1 was conducted. Dendritic cells (DCs): Murine bone-marrow derived DCs were exposed to ALDHi or DMSO and expanded in culture; cells were counted via hematocytometer to assess myeloid cell expansion and DC differentiation. DC vaccine: ALDHi and DMSO were used with a DC vaccine and sonic hedgehog inhibitor in a study of ID8 tumor-bearing mice to assess effects on tumor progression and survival. Results: We identified several novel ALDHi that promote T cell proliferation and CD8 expansion in vitro. Exposure of 5 patient ascites samples to ALDHi showed a significant decrease in CD14+ and CD163+ cells, indicating a loss of M2 immune-suppressive macrophages vs DMSO. NR4A1 expression was decreased in ALDHi treated cells vs DMSO. In contrast, no deleterious effect was observed on ascites T cells. DC expansion: There was a 10-fold increase in DC proliferation when cells were exposed to ALDHi vs DMSO in culture. DC vaccine: In vivo, addition of ALDHi to a DC vaccine did not provide a survival significance. Discussion: In vitro, ALDHi have differential, complementary effects on tumor and immune cells in OC. In tumor cells, ALDHi lower chemoresistance and trigger necroptosis. ALDHi support the expansion of polyclonal CD8 T cells and type-1 DCs and inhibit T-regulatory cells. In contrast, ALDHi reduce M2 macrophages. Despite beneficial effects in vitro, suggesting anti-tumor competence, there was a modest effect on survival in tumor bearing mice. This may be explained by poor bioavailability of current ALDHi. Further analyses of cryopreserved murine tissues (tumor, lymph nodes, spleen) using flow and CHIP cytometry to assess surface markers, Western blot to test levels of NR4A1, and IHC to assess intratumoral immune cell invasion will continue to test target engagement. Alternative in vivo treatment regimens with other compounds are ongoing. Citation Format: Julia Knight, Bingsi Gao, Mainpal Rana, Ibrahim Uygun, Ronald Buckanovich, Anda Vlad. ALDH inhibition as modulator of ovarian tumor associated immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1798.

Phenotypic characterization of disease-initiating stem cells in JAK2- or CALR-mutated myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.

Myeloid Cells As a Promising Target for Brain-Bone Degenerative Diseases from a Metabolic Point of View.

Brain and bone degenerative diseases such as Alzheimer's disease and osteoporosis are common in the aging population and lack efficient pharmacotherapies. Myeloid cells are a diverse group of mononuclear cells that plays important roles in development, immune defense, and tissue homeostasis. Aging drastically alters the expansion and function of myeloid cells, which might be a common pathogenesis of thebrain-bone degenerative diseases. From this perspective, the role of myeloid cells in brain-bone degenerative diseases is discussed, with a particular focus on metabolic alterations in myeloid cells. Furthermore, targeting myeloid cells through metabolic regulation via drugs such as metformin and melatonin is proposed as a potential therapy for the clinical treatment of brain-bone diseases.

Pathogenic role of insufficiency of polycomb repressive complex in primary myelofibrosis

Primary myelofibrosis (PMF) is characterized by the clonal expansion of megakaryocytes and myeloid cells from stem cells with abnormal cytokine expression, resulting in bone marrow fibrosis, angiogenesis, and osteosclerosis. The use of next-generation sequencing revealed that both genetic and epigenetic changes are important in the pathogenesis of PMF. Several epigenetic regulator genes, including TET2, the polycomb-related gene ASXL1, and the polycomb-group gene EZH2, have been found to be targeted by somatic gene mutations in PMF patients. Among these, loss of Ezh2 has been demonstrated to disrupt the function of the polycomb repressive complex 2, promoting the development of JAK2V617F-induced myelofibrosis in mice. In this analysis, we highlight the role of PRC dysfunction in the pathogenesis of PMF.

The Latest Breakthroughs in Immunotherapy for Acute Myeloid Leukemia, with a Special Focus on NKG2D Ligands.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of stem and myeloid progenitor cells. Immunotherapy has revolutionized the care for other cancers such as solid tumors and lymphomas, and has the potential to effectively treat AML. There has been substantial progress in the developments of immunotherapeutic approaches for AML over the last several years, including the development of antibodies that further increase the innate immunogenicity of leukemia cells by the inhibition of NKG2D ligand-particularly MICA and MICB-shedding, chimeric proteins such as IL-15 superagonist that expand natural killer (NK) cells, blockers of immunologic checkpoints such as NKG2A, and chemicals that indirectly increase expression of immune stimulatory proteins in leukemia stem cells. Furthermore, cellular therapies have been designed to enable alloreactive immunity by allogeneic NK cells or target leukemia antigens such as mutated NPM1. These immunotherapeutic approaches have demonstrated remarkable efficacies in preclinical studies and have successfully transitioned to early phase clinical trials, to establish safety and initial signal of clinical activity. Here, we briefly discuss some of the most recent and impactful developments in the AML immunotherapy field and provide our perspectives for the future directions of this exciting and new therapeutic opportunity.

UBTF-TD Expression Is Necessary and Sufficient for Myeloid Cell Expansion

Children with acute myeloid leukemia (AML) have a high relapse rate and dismal overall survival. This can be attributed to a limited number of targeted therapies and a poor understanding of the molecular pathogenesis of AML in childhood. Our group recently identified a subtype of pediatric AML defined by recurrent somatic in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) (Umeda et al., 2022). UBTF-TDs are primarily found in children (~4% of cases at diagnosis), enriched in relapse (~9% of relapse cases), and associated with minimal residual disease (MRD) positivity. UBTF-TD mutations are mutually exclusive with known subtype-defining oncoproteins yet are associated with normal karyotype or trisomy 8 and tend to co-occur with WT1 mutations and FLT3-ITD. Wild-type (WT) UBTF is involved in nucleosome maintenance, chromatin remodeling, and rRNA biogenesis, but the consequences of the tandem duplications on UBTF function, and possibly leukemogenesis, are unknown. We found that expression of UBTF-TD in cord-blood CD34+ (cbCD34+) cells results in increased replating capacity compared to WT-UBTF overexpression as measured by colony forming unit (CFU) assay (UBTF-TD > 4 rounds, WT-UBTF < 2 rounds, q-value < 0.001) as well as increased cellular proliferation. UBTF-TD expression also maintains expression of the stem-cell marker CD117, decreases terminally differentiated myeloid cells (CD11b), and activates the HOXB gene cluster-recapitulating the transcriptional signature observed in patients with UBTF-TD alterations. We, therefore, hypothesized that UBTF-TD expression is a leukemia-initiating event and that leukemogenesis is dependent on UBTF-TDexpression. To assess the leukemic potential of UBTF-TD in vivo, we expressed UBTF-TD in cbCD34+ cells and transplanted them into humanized NSG-SGM3 mice (hSCF hGM-CSF, IL-3) to assess the leukemic potential of UBTF-TD. We observed a rapid in vivo expansion of UBTF-TD expressing cells with an average latency of 11.7 weeks. Peripheral blood immunophenotyping of these mice identified an expansion of myeloid cells (CD33+, CD11b+) and splenomegaly compared to wild-type controls (p-value = 0.0002). These data suggest that UBTF-TDis sufficient to promote myeloid expansion in vivo. To test whether human cbCD34+ myeloid cell expansion is dependent on UBTF-TD expression, we developed an in vitro inducible degradation model by integrating a targetable protein (FKBP12F36V) into our standard lentiviral overexpression vector. This system (dTAG) allows for rapid and direct control of UBTF-TD protein expression. Degradation is achieved by treating these cells with small molecules (i.e., dTAG-13) that recruit the FKBP12F36V-HA-UBTF-TD fusion protein to E3-ubiquitin ligase complex for ubiquitination and subsequent proteasome degradation. Our data shows that the FKBP12F36V tag has no effect on proliferation and replating capacity of UBTF-TD cbCD34+ cells. Treatment with 1 uM dTAG-13 quickly depleted FKBP12F36V-HA-UBTF-TD protein within 1 hour of treatment (fold change when compared to DMSO > 75%); this protein was completely undetectable by western blot after 4hrs. These cells displayed a reduction of CD117 surface marker expression (p-value < 0.0001) as measured by flow cytometry-highlighting a loss in stemness. Furthermore, long-term depletion of UBTF-TD resulted in decreased proliferation (p-value < 0.0001) and cell viability (p-value < 0.0001). This coincided with an increase in apoptosis as measured by Annexin V staining (p-value < 0.0001). Collectively, our data suggest that UBTF-TD expression is sufficient to promote myeloid cell expansion and a leukemic expression profile. This study will help guide therapeutic development in AML as our data shows that directly targeting UBTF-TD for degradation is sufficient to reverse UBTF-TD myeloid cell expansion. Umeda, M., Ma, J., Huang, B. J., Hagiwara, K., Westover, T., Abdelhamed, S., Klco, J. M. (2022). Integrated genomic analysis identifies UBTF tandem duplications as a recurrent lesion in pediatric acute myeloid leukemia. Blood Cancer Discov. doi:10.1158/2643-3230.BCD-21-0160

Traceable Mouse Models for Tracking p53-Mutant Subclones in Acute Myeloid Leukemia

Background: Unleashing the pro-apoptotic effects of p53 by pharmacologic inhibition of MDM2 is an attractive therapeutic strategy in hematologic malignancies with intact p53 1-3. However, concerns were expressed regarding the selection of TP53-mutant subclones with potential GOF activities after treatment with MDM2 inhibitor (MDM2i) 4-8. The emergence of newly detectable TP53 mutations over the course of therapy was also observed in patients receiving intensive chemotherapy 9. Although resistance to treatment leads to selection of TP53-mutant subclones, it is not known whether pre-existing clonal hematopoiesis (CH) sub-clones with TP53 mutation(s) undergo malignant transformation or clonal expansion after exposure to MDM2i therapy. Methods: We generated a traceable Tamoxifen-inducible conditional somatic TP53R172H mutant mouse model to monitor and track the expansion of p53 mutant myeloid cells over the course of therapy with MDM2i. We combined a myeloid-specific Cre line, LysM-CreERT2, as the Cre driver with a conditional knock-in mutant Trp53 allele that constitutively expresses WT Trp53 and converts to mutant (Mut) Trp53R172H upon Cre-mediated recombination (WT to mutant, wm), named Trp53wmR172H13,14. Tamoxifen mediated activation of Cre driven by the myeloid-specific LysM promoter, switches the WTp53 to Mutp53R172H, which corresponds to TP53R175H, a recurrent TP53 mutation in human AML. To trace the recombined hematopoietic cells, we used the Rosa26mTmG reporter allele and performed a pulse-chase experiment in LysM-CreERT2;mTmG;Trp53fx/fx and LysM-CreERT2;mTmG;Trp53R172H/fx mice. Results: Analysis of peripheral blood and the bone marrow (BM) after tamoxifen injection revealed a population of GFP+ myeloid cells, confirming efficient recombination. LysM-CreERT2;mTmG;Trp53R172H/fx mice did not develop leukemia after 1 year upon multiple tamoxifen inductions, suggesting that Trp53 mutation in mature myeloid cells is not leukemogenic. Having a GFP marker in myeloid cells allowed us to monitor the population of GFP+ myeloid cells in peripheral blood after each tamoxifen pulse. The GFP+ cells slowly disappeared from peripheral blood over time and were completely undetectable after 1 month following induction, suggesting that p53 mutation in myeloid cells does not extend the half-life of myeloid cells. Next, to assess if Mdm2 inhibition may cooperate with Trp53 mutation and transform the myeloid cells to AML, we induced the p53 mutation by tamoxifen injection and treated LysM-CreERT2;mTmG;Trp53fx/fx and LysM-CreERT2;mTmG;Trp53R172H/fx mice with the MDM2i, idasanutlin, for 10 days. There were no significant differences in the number of GFP+ myeloid cells between the two groups at baseline, however, in the p53-mutant group, the population of GFP+ cells was significantly decreased and in some mice was fully eradicated after MDM2i treatment. This suggests that p53-mutant protein may have some residual apoptotic activity that is activated after treatment with an MDM2i. In contrast to p53-mutant group, the GFP+ population in the p53-null group did not significantly change in the treated group. Next, we sought to examine if the same response can be achieved in the context of leukemia. Therefore, we transplanted LysM-CreERT2;mTmG;Trp53R172H/fx and control LysM-CreERT2;mTmG;Trp53fx/+ mice with syngeneic MLL-AF9 transformed leukemia cells and treated them with idasanutlin 1 week after transplant. Strikingly, the population of GFP+ cells was highly expanded in LysM-CreERT2;mTmG;Trp53R172H/fx mice transplanted with AML after MDM2i treatment, suggesting that MDM2 inhibition promotes the proliferation of p53-mutant myeloid subclones in the context of AML. To examine if the expanded population was due to transformation of p53-mutant myeloid cells, we sorted the GFP+ cells and transplanted them into syngeneic mice. No leukemia arose after transplantation, suggesting that the expansion of p53-mutant myeloid cells in the context of leukemia was not due to leukemic transformation of p53-mutant cells but rather due to unrestrained proliferation. Conclusion: Overall, these data indicate that the prior existing TP53-mutant subclones are expanded upon idasanutlin treatment without being transformed. Therefore, monitoring of TP53-mutant subclones during the course of treatment is warranted.

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown. Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34+ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34+ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells. To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP+ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia. To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro. In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

Abstract C032: Combination CAF/myeloid targeting in PDAC

Abstract Immunotherapy has revolutionized clinical care for many cancers, yet these treatments fail to control disease in many patients and new strategies are needed to improve anti-tumor immunity and enhance response rates. There is great need for an increased understanding of the cellular crosstalk within tumors and the identification of stromal and immune populations involved in shaping the tumor microenvironment (TME). Local immunosuppression (LIS) is one of the striking hallmarks of Pancreatic Ductal Adenocarcinoma (PDAC), a disease that is highly resistant to existing immunotherapies. Oncogenic Kras activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. LIS is mediated by multiple subtypes of cancer-associated fibroblasts (CAFs) and myeloid cells resident within the tumor parenchyma. Multiple prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the complexity of stromal crosstalk within the TME. The stromal diversity of PDAC complicates investigating paracrine cascades involving multiple cell types. To decipher diverse drug effects on altering the TME, we employ in vivo studies in mouse models recapitulating the human disease, as well as a novel tumor explant model that enables the short-term culture of intact human or murine PDAC. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a delicate balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition (CCR1i) significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C. Hasselluhn, Lukas J. Vlahos, Dafydd Thomas, Alvaro Curiel Garcia, Amanda R. Decker, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Combination CAF/myeloid targeting in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C032.

Bone Marrow Stromal Cell Senescence Induced By Dnmt3a-Mutant Hematopoietic Stem and Progenitor Cells Accelerates Clonal Hematopoiesis and Progression to Leukemia

Clonal hematopoiesis (CH) is commonly driven by mutation in the DNA methyltransferase DNMT3A. While DNMT3A catalyzes DNA methylation, the mechanism(s) by which DNMT3A mutations confer a clonal selective advantage to hematopoietic stem cells (HSCs), and how this can lead to development of acute myeloid leukemia (AML), is not completely understood. Non-hematopoietic cell types in the bone marrow (BM) microenvironment have recently emerged as key contributors in AML by creating a favorable environment for cell growth, including pro-inflammatory changes, accumulation of senescent cells, and the senescence-associated secretory phenotype (SASP). As these alterations have also been found in the context of normal aging, we hypothesized that they contribute to CH-mutant HSC expansion and initiation of AML. To investigate alterations in the BM microenvironment induced by Dnmt3a-mutant hematopoiesis, we transplanted Dnmt3a-mutant or control BM cells into wild-type recipient mice and performed single cell RNA-seq on hematopoietic and non-hematopoietic BM cell fractions. We identified a cellular senescence signature as being enriched in the BM stromal cell (BMSC) subsets Osteo-CAR and Adipo-CAR. We hypothesized that Dnmt3a-mutant hematopoietic stem and progenitor cells (HSPC) remodel the BM microenvironment by induction of senescence to favor their selective advantage. To test this, we developed an ex vivo co-culture system using primary murine BMSC and Dnmt3a-mutant HSPC. Primary BMSCs cultured with Dnmt3a-mutant HSPCs were found to have increased expression of senescence markers (P16, P21, b-galactosidase) and SASP (IL-6, IL-1a) compared to BMSC cultured with control wild-type (WT) HSPCs. This phenotype was similarly observed in BMSCs in vivo after Dnmt3a-mutant cells were transplanted into WT recipient mice, in addition to increased expression of the anti-apoptotic proteins BCL-2 and BCL-xL in BMSCs. We then pursued mechanistic experiments to reveal how Dnmt3a-mutant HSPCs induce senescence of BMSCs. Transwell assays supported that senescence induction was cell contact independent. Cytokine profiling of conditioned media from Dnmt3a-mutant HSPCs, and well as BM fluid of transplanted recipient mice, revealed elevated levels of the inflammatory cytokines IL-6, S100A8/9 and TNFα. We directly tested the effect of IL-6 on primary BMSC by culture with recombinant IL-6 alone and found that this was sufficient to increase b-galactosidase staining in BMSCs. To determine the extent to which deletion of senescent cells can abrogate Dnmt3a-mutant selective advantage and progression to hematologic malignancy, we used our inducible mouse model of Dnmt3a;Npm1-mutant AML (Loberg et al., Leukemia 2019). We transplanted cells carrying the Dnmt3a mutation into WT recipient mice, administered the senolytic Navitoclax or vehicle control, and then induced Npm1 mutation in vivo. We found that Navitoclax-treated mice had reduced peripheral blood myeloid cell expansion, a biomarker of Dnmt3a;Npm1-mutant transformation. Furthermore, Navitoclax-treated mice had reduced WBC and monocyte counts, reduced bone marrow engraftment, reduced myeloid colony-forming units (CFU), and maintained lower BMSC senescence. Together, we report that Dnmt3a-mutant HSPCs induce senescence of BMSCs through production of the pro-inflammatory cytokine IL-6. Depletion of these senescent cells using Navitoclax reduces myeloproliferation and transformation of Dnmt3a-mutant HSPCs to AML. We suggest that SASP may be used as a biomarker of transformation risk in individuals with CH, and that targeting the induced senescent BM microenvironment may be used prophylactically to prevent transformation to AML.

EXTH-70. IMMUNOTHERAPY REVERSES GLIOMA-DRIVEN DYSFUNCTION OF IMMUNE SYSTEM HOMEOSTASIS

Abstract BACKGROUND Glioma-induced immune disfunction has been described in a limited number of studies, and here we further demonstrate that gliomas also interrupt the cellular differentiation programming and outcomes of hematopoietic stem cells (HSC) in the bone marrow. HSCs from glioma-bearing mice are re-programmed and driven towards expansion of myeloid lineage precursors myeloid-derived suppressor cells (MDSC) in secondary lymphoid organs. However, we found this is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of CNS malignancies, and here we describe how glioma-induced disfunction is reversed by this immunotherapeutic platform. METHODS The impact of orthotopic KR158B glioma on HSCs was evaluated in an unbiased fashion using single cell RNAseq of lineage- cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor-specific dendritic cells, tumor-reactive T cells, and HSCs isolated from glioma-bearing or non-tumor-bearing mice was used to evaluate cell fate differentiation and survival. RESULTS We observed differential gene expression of HSCs in tumor-bearing versus healthy mice coupled with an expansion of myeloid lineage subsets in glioma-bearing mice. Interestingly, MDSCs from glioma-bearing mice demonstrated hyper suppressive capacity as compared to MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame the suppressive properties of glioma-bearing HSCs. When HSCs from tumor-bearing mice were transferred with ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs. CONCLUSIONS These findings demonstrate the suppressive disposition in HSCs from glioma-bearing hosts can be redirected using ACT for significant survival benefit. Collectively, these data demonstrate gliomas impact the hematopoietic compartment at the progenitor level to promote expansion of suppressive myeloid cells and their progenitors. However, ACT can overcome this bias and provide lasting anti-tumor effects.

Therapeutic activity of GARP:TGF-β1 blockade in murine primary myelofibrosis

AbstractPrimary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-β1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-β1, only a few activate the cytokine via cell type–specific mechanisms. The cellular source of the active TGF-β1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-β1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-β1 activation by GARP-expressing cells. GARP:TGF-β1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-β1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-β1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

The cytokine network in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous malignancy of the blood and bone marrow, characterized by clonal expansion of myeloid stem and progenitor cells and rapid disease progression. Chemotherapy has been the first-line treatment for AML for more than 30 years. Application of recent high-throughput next-generation sequencing technologies has revealed significant molecular heterogeneity to AML, which in turn has motivated efforts to develop new, targeted therapies. However, due to the high complexity of this disease, including multiple driver mutations and the coexistence of multiple competing tumorigenic clones, the successful incorporation of these new agents into clinical practice remains challenging. These continuing difficulties call for the identification of innovative therapeutic approaches that are effective for a larger cohort of AML patients. Recent studies suggest that chronic immune stimulation and aberrant cytokine signaling act as triggers for AML initiation and progression, facets of the disease which might be exploited as promising targets in AML treatment. However, despite the greater appreciation of cytokine profiles in AML, the exact functions of cytokines in AML pathogenesis are not fully understood. Therefore, unravelling the molecular basis of the complex cytokine networks in AML is a prerequisite to develop new therapeutic alternatives based on targeting cytokines and their receptors.