Oncogenic dependency on SWI/SNF chromatin remodeling factors in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy arising from immature thymocytes. Unlike well-known oncogenic transcription factors, such as NOTCH1 and MYC, the involvement of chromatin remodeling factors in T-ALL pathogenesis is poorly understood. Here, we provide compelling evidence on how SWI/SNF chromatin remodeling complex contributes to human T-ALL pathogenesis. Integrative analysis of transcriptomic and ATAC-Seq datasets revealed high expression of SMARCA4, one of the subunits of the SWI/SNF complex, in T-ALL patient samples and cell lines compared to normal T cells. Loss of SMARCA protein function resulted in apoptosis induction and growth inhibition in multiple T-ALL cell lines. ATAC-Seq analysis revealed a massive reduction in chromatin accessibility across the genome after the loss of SMARCA protein function. RUNX1 interacts with SMARCA4 protein and co-occupies the same genomic regions. Importantly, the NOTCH1-MYC pathway was primarily affected when SMARCA protein function was impaired, implicating SWI/SNF as a novel therapeutic target.

Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

The role of quiescent thymic progenitors in TAL/LMO2-induced T-ALL chemotolerance.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

ZBP1 activation triggers hematopoietic stem and progenitor cell death resulting in bone marrow failure in mice.

Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.

TBK1 inhibition unleashes RIPK1, resensitizing tumors to immunotherapy.

Resistance mechanisms have curbed the potential of immune checkpoint blockade (ICB) therapies. Understanding mechanisms that contribute to this resistance should reveal new targets for combinatorial therapy. Tank-binding kinase 1 (TBK1) represents such a target. In recent work by Sun et al., inhibition of TBK1 restored the efficacy of such treatments by sensitizing tumors to RIPK1 kinase-dependent inflammatory cell death.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

ESRRB regulates glucocorticoid gene expression in mice and patients with acute lymphoblastic leukemia.

Synthetic glucocorticoids (GCs), such as dexamethasone and prednisone, remain key components of therapy for patients with lymphoid malignancies. For pediatric patients with acute lymphoblastic leukemia (ALL), response to GCs remains the most reliable prognostic indicator; failure to respond to GC correlates with poor event-free survival. To uncover GC resistance mechanisms, we performed a genome-wide, survival-based short hairpin RNA screen and identified the orphan nuclear receptor estrogen-related receptor-ß (ESRRB) as a critical transcription factor that cooperates with the GC receptor (GR) to mediate the GC gene expression signature in mouse and human ALL cells. Esrrb knockdown interfered with the expression of genes that were induced and repressed by GR and resulted in GC resistance in vitro and in vivo. Dexamethasone treatment stimulated ESRRB binding to estrogen-related receptor elements (ERREs) in canonical GC-regulated genes, and H3K27Ac Hi-chromatin immunoprecipitation revealed increased interactions between GR- and ERRE-containing regulatory regions in dexamethasone-treated human T-ALL cells. Furthermore, ESRRB agonists enhanced GC target gene expression and synergized with dexamethasone to induce leukemic cell death, indicating that ESRRB agonists may overcome GC resistance in ALL, and potentially, in other lymphoid malignancies.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

RIPK1 Mediates TNF-Induced Intestinal Crypt Apoptosis During Chronic NF-κB Activation.

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice. METHODS: Human IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKß in IEC (Ikkß(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation. RESULTS: NF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkß(EE)IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKß facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo. CONCLUSIONS: Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD.

NOTCH1 Represses MCL-1 Levels in GSI-resistant T-ALL, Making them Susceptible to ABT-263.

PURPOSE: Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models. RESULTS: We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo. CONCLUSIONS: Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

Elevated A20 promotes TNF-induced and RIPK1-dependent intestinal epithelial cell death.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

Analyzing Necroptosis Using an RIPK1 Kinase Inactive Mouse Model of TNF Shock.

The serine/threonine kinase RIPK1 has numerous biological and pathological functions, mediating prosurvival as well as prodeath apoptotic and necroptotic signaling pathways downstream of various receptors, including death receptors and Toll-like receptors (TLRs). RIPK1 has been implicated in various diseases, including ischemia-reperfusion injury and inflammatory bowel disease (IBD). The recent generation of RIPK1 kinase inactive mice has enabled us to genetically interrogate the role of RIPK1 kinase-mediated necroptosis in disease models. Here, we describe procedures utilizing kinase inactive Ripk1D138N/D138N mice to analyze necroptosis induction in vitro in bone-marrow derived macrophages (BMDMs) and in vivo in a murine model of TNF-induced shock.

Oncogenic hijacking of the stress response machinery in T cell acute lymphoblastic leukemia.

Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress response machinery by inducing the expression of HSF1 and its downstream effectors. The NOTCH1 signaling status controls the levels of chaperone/co-chaperone complexes and predicts the response of T-ALL patient samples to HSP90 inhibition. Our data demonstrate an integral crosstalk between mediators of oncogene and non-oncogene addiction and reveal critical nodes of the heat shock response pathway that can be targeted therapeutically.

RIP kinase 1-dependent endothelial necroptosis underlies systemic inflammatory response syndrome.

Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context-dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory "cytokine storm" has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase-inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.

NOTCH Signaling in T-Cell-Mediated Anti-Tumor Immunity and T-Cell-Based Immunotherapies.

The NOTCH (1-4) family of receptors are highly conserved and are critical in regulating many developmental processes and in the maintenance of tissue homeostasis. Our laboratory and numerous others have demonstrated that aberrant NOTCH signaling is oncogenic in several different cancer types. Conversely, there is also evidence that NOTCH can also function as a tumor suppressor. In addition to playing an essential role in tumor development, NOTCH receptors regulate T-cell development, maintenance, and activation. Recent studies have determined that NOTCH signaling is required for optimal T-cell-mediated anti-tumor immunity. Consequently, tumor cells and the tumor microenvironment have acquired mechanisms to suppress NOTCH signaling to evade T-cell-mediated killing. Tumor-mediated suppression of NOTCH signaling in T-cells can be overcome by systemic administration of NOTCH agonistic antibodies and ligands or proteasome inhibitors, resulting in sustained NOTCH signaling and T-cell activation. In addition, NOTCH receptors and ligands are being utilized to improve the generation and specificity of T-cells for adoptive transplant immunotherapies. In this review, we will summarize the role(s) of NOTCH signaling in T-cell anti-tumor immunity as well as TCR- and chimeric antigen receptor-based immunotherapies.

Dendritic Cell RIPK1 Maintains Immune Homeostasis by Preventing Inflammation and Autoimmunity.

Necroptosis is a form of cell death associated with inflammation; however, the biological consequences of chronic necroptosis are unknown. Necroptosis is mediated by RIPK1, RIPK3, and MLKL kinases but in hematopoietic cells RIPK1 has anti-inflammatory roles and functions to prevent necroptosis. Here we interrogate the consequences of chronic necroptosis on immune homeostasis by deleting Ripk1 in mouse dendritic cells. We demonstrate that deregulated necroptosis results in systemic inflammation, tissue fibrosis, and autoimmunity. We show that inflammation and autoimmunity are prevented upon expression of kinase inactive RIPK1 or deletion of RIPK3 or MLKL. We provide evidence that the inflammation is not driven by microbial ligands, but depends on the release of danger-associated molecular patterns and MyD88-dependent signaling. Importantly, although the inflammation is independent of type I IFN and the nucleic acid sensing TLRs, blocking these pathways rescues the autoimmunity. These mouse genetic studies reveal that chronic necroptosis may underlie human fibrotic and autoimmune disorders.