Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

Ubiquitination of hnRNPA1 by TRAF6 links chronic innate immune signaling with myelodysplasia.

Toll-like receptor (TLR) activation contributes to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS). TRAF6, a TLR effector with ubiquitin (Ub) ligase activity, is overexpressed in MDS hematopoietic stem/progenitor cells (HSPCs). We found that TRAF6 overexpression in mouse HSPC results in impaired hematopoiesis and bone marrow failure. Using a global Ub screen, we identified hnRNPA1, an RNA-binding protein and auxiliary splicing factor, as a substrate of TRAF6. TRAF6 ubiquitination of hnRNPA1 regulated alternative splicing of Arhgap1, which resulted in activation of the GTP-binding Rho family protein Cdc42 and accounted for hematopoietic defects in TRAF6-expressing HSPCs. These results implicate Ub signaling in coordinating RNA processing by TLR pathways during an immune response and in premalignant hematologic diseases, such as MDS.

In vivo ablation of NF-κB cascade effectors alleviates disease burden in myeloproliferative neoplasms.

ABSTRACT: Hyperactivation of the NF-κB cascade propagates oncogenic signaling and proinflammation, which together augments disease burden in myeloproliferative neoplasms (MPNs). Here, we systematically ablate NF-κB signaling effectors to identify core dependencies using a series of primary samples and syngeneic and patient-derived xenograft (PDX) mouse models. Conditional knockout of Rela attenuated Jak2V617F- and MPLW515L-driven onset of polycythemia vera and myelofibrosis disease hallmarks, respectively. In PDXs, RELA knockout diminished leukemic engraftment and bone marrow fibrosis while extending survival. Knockout of upstream effector Myd88 also alleviated disease burden; conversely, perturbation of negative regulator miR-146a microRNA induced earlier lethality and exacerbated disease. Perturbation of NF-κB effectors further skewed the abundance and distribution of hematopoietic multipotent progenitors. Finally, pharmacological targeting of interleukin-1 receptor-associated kinase 4 (IRAK4) with inhibitor CA-4948 suppressed disease burden and inflammatory cytokines specifically in MPN without inducing toxicity in nondiseased models. These findings highlight vulnerabilities in MPN that are exploitable with emerging therapeutic approaches.

Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML.

Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in preclinical studies and clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. In this study, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that cotargeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. Although IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complementary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via noncanonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the polycomb repressive complex 2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in xenograft and in vitro studies when treated with KME-2780 as compared with selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for cotargeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

IRAK1 and IRAK4 as emerging therapeutic targets in hematologic malignancies.

PURPOSE OF REVIEW: Cell intrinsic and extrinsic perturbations to inflammatory signaling pathways are a hallmark of development and progression of hematologic malignancies. The interleukin 1 receptor-associated kinases (IRAKs) are a family of related signaling intermediates (IRAK1, IRAK2, IRAK3, IRAK4) that operate at the nexus of multiple inflammatory pathways implicated in the hematologic malignancies. In this review, we explicate the oncogenic role of these kinases and review recent therapeutic advances in the dawning era of IRAK-targeted therapy. RECENT FINDINGS: Emerging evidence places IRAK signaling at the confluence of adaptive resistance and oncogenesis in the hematologic malignancies and solid tissue tumors. Preclinical investigations nominate the IRAK kinases as targetable molecular dependencies in diverse cancers. SUMMARY: IRAK-targeted therapies that have matriculated to early phase trials are yielding promising preliminary results. However, studies of IRAK kinase signaling continue to defy conventional signaling models and raise questions as to the design of optimal treatment strategies. Efforts to refine IRAK signaling mechanisms in the malignant context will inspire deliberate IRAK-targeted drug development and informed combination therapy.

Chronic immune response dysregulation in MDS pathogenesis.

Chronic innate immune signaling in hematopoietic cells is widely described in myelodysplastic syndromes (MDS), and innate immune pathway activation, predominantly via pattern recognition receptors, increases the risk of developing MDS. An inflammatory component to MDS has been reported for many years, but only recently has evidence supported a more direct role of chronic innate immune signaling and associated inflammatory pathways in the pathogenesis of MDS. Here we review recent findings and discuss relevant questions related to chronic immune response dysregulation in MDS.

Inhibition of IRAK1 Ubiquitination Determines Glucocorticoid Sensitivity for TLR9-Induced Inflammation in Macrophages.

Inflammatory responses are controlled by signaling mediators that are regulated by various posttranslational modifications. Recently, transcription-independent functions for glucocorticoids (GC) in restraining inflammation have emerged, but the underlying mechanisms are unknown. In this study, we report that GC receptor (GR)-mediated actions of GC acutely suppress TLR9-induced inflammation via inhibition of IL-1R-associated kinase 1 (IRAK1) ubiquitination. ß-TrCP-IRAK1 interaction is required for K48-linked ubiquitination of IRAK1 at Lys134 and subsequent membrane-to-cytoplasm trafficking of IRAK1 interacting partners TNFR-associated factor 6 and TAK1 that facilitates NF-κB and MAPK activation. Upon costimulation of macrophages with GC and TLR9-engaging ligand, GR physically interacts with IRAK1 and interferes with protein-protein interactions between ß-TrCP and IRAK1. Ablation of GR in macrophages prevents GC-dependent suppression of ß-TrCP-IRAK1 interactions. This GC-mediated suppression of IRAK1 activation is unique to TLR9, as GC treatment impairs TLR9 but not TLR4 ligand-induced K48-linked IRAK1 ubiquitination and trafficking of IRAK1 interacting partners. Furthermore, mutations in IRAK1 at Lys134 prevent TLR9 ligand-induced activation of inflammatory signaling mediators and synthesis of proinflammatory cytokines to an extent comparable to GC-mediated inhibition. Collectively, these findings identify a transcription-independent, rapid, and nongenomic GC suppression of TLR9 ligand-mediated IRAK1 ubiquitination as a novel mechanism for restraining acute inflammatory reactions.

Errant innate immune signaling in del(5q) MDS.

In this issue of Blood, Keerthivasan et al have identified that the deletion of mDia1, a chromosome 5q gene, contributes to myelodysplastic syndromes (MDSs) by increasing innate immune signaling in granulocytes.

Cytotoxic effects of bortezomib in myelodysplastic syndrome/acute myeloid leukemia depend on autophagy-mediated lysosomal degradation of TRAF6 and repression of PSMA1.

Bortezomib (Velcade) is used widely for the treatment of various human cancers; however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that bortezomib induces proteasome-independent degradation of the TRAF6 protein, but not mRNA, in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of bortezomib due to down-regulation of the proteasomal subunit α-1 (PSMA1). To determine the molecular consequences of loss of TRAF6 in MDS/AML cells, in the present study, we applied gene-expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe herein novel mechanisms by which TRAF6 is regulated through bortezomib/autophagy-mediated degradation and by which it alters MDS/AML sensitivity to bortezomib by controlling PSMA1 expression.

DDX41 haploinsufficiency causes inefficient hematopoiesis under stress and cooperates with p53 mutations to cause hematologic malignancy.

Germline heterozygous mutations in DDX41 predispose individuals to hematologic malignancies in adulthood. Most of these DDX41 mutations result in a truncated protein, leading to loss of protein function. To investigate the impact of these mutations on hematopoiesis, we generated mice with hematopoietic-specific knockout of one Ddx41 allele. Under normal steady-state conditions, there was minimal effect on lifelong hematopoiesis, resulting in a mild yet persistent reduction in red blood cell counts. However, stress induced by transplantation of the Ddx41+/- BM resulted in hematopoietic stem/progenitor cell (HSPC) defects and onset of hematopoietic failure upon aging. Transcriptomic analysis of HSPC subsets from the transplanted BM revealed activation of cellular stress responses, including upregulation of p53 target genes in erythroid progenitors. To understand how the loss of p53 affects the phenotype of Ddx41+/- HSPCs, we generated mice with combined Ddx41 and Trp53 heterozygous deletions. The reduction in p53 expression rescued the fitness defects in HSPC caused by Ddx41 heterozygosity. However, the combined Ddx41 and Trp53 mutant mice were prone to developing hematologic malignancies that resemble human myelodysplastic syndrome and acute myeloid leukemia. In conclusion, DDX41 heterozygosity causes dysregulation of the response to hematopoietic stress, which increases the risk of transformation with a p53 mutation.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression.

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

Dysregulated innate immune signaling cooperates with RUNX1 mutations to transform an MDS-like disease to AML.

Dysregulated innate immune signaling is linked to preleukemic conditions and myeloid malignancies. However, it is unknown whether sustained innate immune signaling contributes to malignant transformation. Here we show that cell-intrinsic innate immune signaling driven by miR-146a deletion (miR-146aKO), a commonly deleted gene in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), cooperates with mutant RUNX1 (RUNX1mut) to initially induce marrow failure and features of MDS. However, miR-146aKO hematopoietic stem and/or progenitor cells (HSPCs) expressing RUNX1mut eventually progress to a fatal AML. miR-146aKO HSPCs exhaust during serial transplantation, while expression of RUNX1mut restored their hematopoietic cell function. Thus, HSPCs exhibiting dysregulated innate immune signaling require a second hit to develop AML. Inhibiting the dysregulated innate immune pathways with a TRAF6-UBE2N inhibitor suppressed leukemic miR-146aKO/RUNX1mut HSPCs, highlighting the necessity of TRAF6-dependent cell-intrinsic innate immune signaling in initiating and maintaining AML. These findings underscore the critical role of dysregulated cell-intrinsic innate immune signaling in driving preleukemic cells toward AML progression.

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation.

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations.

Genome-wide identification of human microRNAs located in leukemia-associated genomic alterations.

Cytogenetic alterations, such as amplifications, deletions, or translocations, contribute to myeloid malignancies. MicroRNAs (miRNAs) have emerged as critical regulators of hematopoiesis, and their aberrant expression has been associated with leukemia. Genomic regions containing sequence alterations and fragile sites in cancers are enriched with miRNAs; however, the relevant miRNAs within these regions have not been evaluated on a global basis. Here, we investigated miRNAs relevant to acute myeloid leukemia (AML) by (1) mapping miRNAs within leukemia-associated genomic alterations in human AML cell lines by high-resolution genome arrays and (2) evaluating absolute expression of these miRNAs by massively parallel small RNA sequencing. Seventy-seven percent (542 of 706) of miRNAs mapped to leukemia-associated copy-number alterations in the cell lines; however, only 18% (99 of 542) of these miRNAs are expressed above background levels. As evidence that this subset of miRNAs is relevant to leukemia, we show that loss of 2 miRNAs identified in our analysis, miR-145 and miR-146a, results in leukemia in a mouse model. Small RNA sequencing identified 28 putative novel miRNAs, 18 of which map to leukemia-associated copy-number alterations. This detailed genomic and small RNA analysis points to a subset of miRNAs that may play a role in myeloid malignancies.

Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells.

Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.