Lyndsey Bolanos

Targeting IRAK1 as a Therapeutic Approach for Myelodysplastic Syndrome

Myelodysplastic syndromes (MDSs) arise from a defective hematopoietic stem/progenitor cell. Consequently, there is an urgent need to develop targeted therapies capable of eliminating the MDS-initiating clones. We identified that IRAK1, an immune-modulating kinase, is overexpressed and hyperactivated in MDSs. MDS clones treated with a small molecule IRAK1 inhibitor (IRAK1/4-Inh) exhibited impaired expansion and increased apoptosis, which coincided with TRAF6/NF-κB inhibition. Suppression of IRAK1, either by RNAi or with IRAK1/4-Inh, is detrimental to MDS cells, while sparing normal CD34(+) cells. Based on an integrative gene expression analysis, we combined IRAK1 and BCL2 inhibitors and found that cotreatment more effectively eliminated MDS clones. In summary, these findings implicate IRAK1 as a drugable target in MDSs.

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.

Myeloid malignancies with chromosome 5q deletions acquire a dependency on an intrachromosomal NF-κB gene network.

Chromosome 5q deletions (del[5q]) are common in high-risk (HR) myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); however, the gene regulatory networks that sustain these aggressive diseases are unknown. Reduced miR-146a expression in del(5q) HR MDS/AML and miR-146a(-/-) hematopoietic stem/progenitor cells (HSPCs) results in TRAF6/NF-κB activation. Increased survival and proliferation of HSPCs from miR-146a(low) HR MDS/AML is sustained by a neighboring haploid gene, SQSTM1 (p62), expressed from the intact 5q allele. Overexpression of p62 from the intact allele occurs through NF-κB-dependent feedforward signaling mediated by miR-146a deficiency. p62 is necessary for TRAF6-mediated NF-κB signaling, as disrupting the p62-TRAF6 signaling complex results in cell-cycle arrest and apoptosis of MDS/AML cells. Thus, del(5q) HR MDS/AML employs an intrachromosomal gene network involving loss of miR-146a and haploid overexpression of p62 via NF-κB to sustain TRAF6/NF-κB signaling for cell survival and proliferation. Interfering with the p62-TRAF6 signaling complex represents a therapeutic option in miR-146a-deficient and aggressive del(5q) MDS/AML.

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.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 (HSPC). Here we show that TRAF6 overexpression in mouse HSPC resulted in impaired hematopoiesis and bone marrow failure. Through the use of 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 Cdc42 activation and accounted for hematopoietic defects in TRAF6-expressing HSPC. These results implicate Ub signaling in coordinating RNA processing by TLR pathways during an immune response and in premalignant hematologic diseases, such as MDS.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.

A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival

The chromatin-binding DEK protein was recently reported to promote the growth of HPV+ and HPV− head and neck squamous cell carcinomas (HNSCCs). Relevant cellular and molecular mechanism(s) controlled by DEK in HNSCC remain poorly understood. While DEK is known to regulate specific transcriptional targets, global DEK-dependent gene networks in HNSCC are unknown. To identify DEK transcriptional signatures we performed RNA-Sequencing (RNA-Seq) in HNSCC cell lines that were either proficient or deficient for DEK. Bioinformatic analyses and subsequent validation revealed that IRAK1, a regulator of inflammatory signaling, and IRAK1-dependent regulatory networks were significantly repressed upon DEK knockdown in HNSCC. According to TCGA data, 14% of HNSCC specimens overexpressed IRAK1, thus supporting possible oncogenic functions. Furthermore, genetic or pharmacologic inhibition of IRAK1 in HNSCC cell lines was sufficient to attenuate downstream signaling such as ERK1/2 and to induce HNSCC cell death by apoptosis. Finally, targeting DEK and IRAK1 simultaneously enhanced cell death as compared to targeting either alone. Our findings reveal that IRAK1 promotes cell survival and is an attractive therapeutic target in HNSCC cells. Thus, we propose a model wherein IRAK1 stimulates tumor signaling and phenotypes both independently and in conjunction with DEK.

Differential IRAK signaling in hematologic malignancies

Interleukin receptor-associated kinase (IRAK) family mediates signals downstream of various pathogen- and cytokine-responsive receptors [1,2]. IRAK proteins consist of four functionally and structurally related members (IRAK1–4). In the context of hematologic disorders, IRAK1 and IRAK4 are the most widely studied [3], and are both ubiquitously expressed [4]. Under normal cellular conditions, MyD88 is recruited to activated Toll-like receptors (TLRs) or Inter-leukin 1 receptor (IL1R) resulting in activation of IRAK4 and IRAK1 [5]. Activated IRAK1/4 proteins then bind TRAF6 mediating NF-κB signaling. Activating mutations of MyD88 or B cell receptor result in chronic IRAK4 phosphorylation and downstream pathway activation in human B cell lymphoma, particularly in the activated B cell–like (ABC) subset of diffuse large B cell lymphoma (DLBCL) [3,6]. Knockdown of MyD88, IRAK4, or IRAK1 abrogates NF-κB pathway activation and induces ABC DLBCL cell death [3]. Interestingly, IRAK4 catalytic function is necessary for maintaining the viability of DLBCL cells, whereas the catalytic function of IRAK1 is dispensable [3]. These critical observations strongly implicate the dependency of ABC DLBCL on IRAK4 function. More recently, we have reported that IRAK1 exists in an activated state (e.g., constitutively phosphorylated on threonine-209) in a large subset of human myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) samples [7]. In addition, overexpression of TLR1/2/6 has been reported in MDS, and MDS-associated mutations of TLR2 correspond with increased IRAK1 activation [8]. MDS originates within the hematopoietic stem cell compartment and manifests into a multilineage erythro/myeloid disease [9]. Patients with MDS also have a proclivity to develop AML [9]. Knockdown of IRAK1 in MDS marrow cells and in a panel of MDS/AML cell lines resulted in cell cycle arrest, apoptosis, and impaired leukemic progenitor function. To further validate these findings, we treated cells with an IRAK1/4 Inhibitor. Consistent with the knockdown experiments, IRAK1/4 Inhibitor impaired MDS/AML cell viability and progenitor function, which also coincided with reduced levels of phosphorylated IRAK1, but not IRAK4. Given the importance of the IRAK1/IRAK4 complex in human hematologic malignancies, we decided to investigate the role of IRAK4 in MDS. To discern differences between the expression of IRAK1 and IRAK4, published microarray data from MDS CD34+ cells were examined [10]. IRAK4 expression is extremely low (at the lower limit of detection) and not significantly different as compared with control CD34+ cells (p = 0.073; Figure 1). By comparison, IRAK1 is preferentially expressed in normal CD34+ cells and further overexpressed in a subset (~20%) of MDS patients (p = 0.036; Figure 1). To evaluate the contribution of IRAK1 versus IRAK4 in MDS cells functionally, we performed RNAi-mediated knockdown experiments. An MDS cell line (MDSL) transduced with shRNA targeting IRAK1 or IRAK4 were first evaluated for RNA and protein knockdown. As shown in Figures 1B and C, shIRAK1 clone #17 and shIRAK4 clone #65 resulted in approximately 90% knockdown of the respected targets; therefore, these shRNA clones were selected for further validation. As described extensively in our recent report [7], knockdown of IRAK1 in MDSL cells results in apoptosis (Figure 1D) and impaired progenitor function in methylcellulose (Figure 1E). In contrast, knockdown of IRAK4 did not contribute to significant cell death of MDS cells (Figure 1D). Furthermore, knockdown of IRAK4 in MDSL reduced progenitor function (p = 0.0013), but not as dramatically as seen with knockdown of IRAK1 (p = 0.0004; Figure 1E). Under stimulated conditions or in DLBCL, IRAK4 phosphorylates IRAK1. Interestingly, knockdown of IRAK4 in MDS cells negligibly affects phosphorylated IRAK1 levels (Figure 1F), suggesting that IRAK1 is activated by alternative mechanisms in MDS. These findings reveal differences in IRAK1 versus IRAK4 dependency in MDS. Figure 1 MDS cells depend on IRAK1, but less on IRAK4, for maintaining viability and progenitor function. (A) IRAK1 and IRAK4 expression is obtained from a gene expression study on CD34+ cells isolated from control and MDS marrows (IRAK1; p = 0.036) [10]. (B) ... IRAK1 and IRAK4 are related kinases within the innate immune pathway. However, their roles in myeloid versus lymphoid malignancies appear to be distinct. The work by Staudt and colleagues has clearly established a critical role of IRAK4 in DLBCL and the efficacy of targeting IRAK4 using an IRAK1/4 inhibitor [3]. We propose that IRAK1, but not IRAK4, is essential in the pathogenesis of MDS/AML, and that small molecules selectively targeting IRAK1 may be therapeutically beneficial in MDS/AML. In conclusion, the innate immune pathway involving IRAK1 and IRAK4 signaling is important in the pathogenesis of hematologic malignancies, but their individual contribution is lineage or disease specific, or both. As such, further research to discern the individual contribution of IRAK1 and IRAK4 to hematologic malignancies is warranted. Nevertheless, development of next generation IRAK inhibitors could be beneficial in both myeloid and lymphoid malignancies.

Epistasis between TIFAB and miR-146a: neighboring genes in del(5q) myelodysplastic syndrome

Epistasis between TIFAB and miR-146a: neighboring genes in del(5q) myelodysplastic syndrome

TRAF6 Mediates Basal Activation of NF-κB Necessary for Hematopoietic Stem Cell Homeostasis

SUMMARY Basal nuclear factor κB (NF-κB) activation is required for hematopoietic stem cell (HSC) homeostasis in the absence of inflammation; however, the upstream mediators of basal NF-κB signaling are less well understood. Here, we describe TRAF6 as an essential regulator of HSC homeostasis through basal activation of NF-κB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient hematopoietic stem/progenitor cells (HSPCs) revealed changes in adaptive immune signaling, innate immune signaling, and NF-κB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection is required for HSC function. In addition, we established that loss of IκB kinase beta (IKKβ)- mediated NF-κB activation is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKβ similarly resulted in impaired HSC self-renewal and fitness. Taken together, TRAF6 is required for HSC homeostasis by maintaining a minimal threshold level of IKKβ/NF-κB signaling.