Cytokine Receptor-like Factor Research Articles

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

Abstract B134: Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk hematological malignancy with poor outcome, and new treatments are urgently required. We address this challenge by targeting cytokine receptor-like factor 2 (CRLF2), which is overexpressed in ~50% of Ph-like ALL cases conferring chemoresistance and survival of Ph-like ALL cells through JAK/STAT signaling. CRLF2 is an ideal target due to its role in leukemogenesis, limiting the likelihood of relapse associated with antigen loss, and its minimal expression in healthy tissue. Here, we developed a novel CRLF2-targeting antibody fragment-drug conjugate (CRLF2-FDC) and evaluated its efficacy in preclinical models. Methods: A single-chain anti-CRLF2 variable monoclonal antibody fragment (scFv-Fc) was engineered by conventional hybridoma technology, PCR and expression in HEK293T cells. The scFv-Fc was conjugated to the anti-microtubule drug DM1 via a non-cleavable linker. The selective association and internalization of the CRLF2-FDC was determined in vitrousing Ph-like ALL cell lines and their CRLF2-knockdown counterparts, and ex vivo using patient-derived xenograft (PDX) cells. The in vivo efficacy of the CRLF2-FDC (equivalent to 10 mg/kg scFv-Fc, 0.4 mg/kg DM1, once weekly x 4 weeks via intravenous injection) was tested against pediatric ALL PDX models in immune-deficient (NSG) mice, in comparison to scFv-Fc or DM1 alone. The efficacy of the CRLF2-FDC was further evaluated following two weeks of standard-of-care induction chemotherapy [vincristine (0.15 mg/kg, once weekly), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off (VXL)]. Treatment efficiency was calculated by subtracting the median event-free survival (EFS) of the control and treated groups (T-C) and stringent objective response criteria (progressive disease, PD; partial response, PR; complete response, CR; maintained CR, MCR) and leukemia infiltration in major organs at day 28 post treatment initiation. Results: CRLF2-dependent association (P≤0.01) and internalization of the CRLF2-FDC was confirmed both in vitro and in vivo in Ph-like ALL cells. While the scFv-Fc alone or free DM1 elicited PDs in a Ph-like ALL PDX in vivo (T-C 6 and 1.6 days, respectively, P=0.0013 for both), the CRLF2-FDC achieved a PR (T-C 32 days, P=0.0088). Besides targeted drug delivery, the CRLF2-FDC also demonstrated a significant antagonistic impact on STAT5 signaling in vivo 8 h after administration, suggesting a two-pronged therapeutic approach. Notably, the CRLF2-FDC achieved an MCR when administered after VXL (T-C 53.9 days, P <0.0001) compared to a CR (T-C 25.2 days, P <0.0001) by VXL alone. Furthermore, the CRLF2-FDC significantly decreased leukemic burden in major organs at 28 days post treatment initiation. Conclusions: This study describes the selective potency and promising preclinical activity of a novel CRLF2-FDC for the treatment of Ph-like ALL when administered either as a single agent or following standard-of-care induction therapy. Citation Format: Richard B Lock, Sara M.A. Mohamed, Peter Schofield, Karl-Heinz Friedrich, Daniel Christ, Maria Kavallaris, Narges Bayat. Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B134.

CRLF2 Expression in Mexicans with Acute Lymphoblastic Leukemia across the Age Spectrum

Introduction Acute lymphoblastic leukemia (ALL) is a hematological malignancy that primarily affects children also occurs in adults. Certain subtypes of ALL, such as Philadelphia chromosome-like ALL (Ph-like ALL), present unique challenges and are associated with poor outcomes. Alterations in the CRLF2 (Cytokine Receptor-like Factor 2) gene have emerged as critical contributors to the pathogenesis of Ph-like ALL. CRLF2 gene rearrangements result in the overexpression of CRLF2 protein, leading to dysregulated signaling pathways and enhanced leukemogenesis. The prevalence and clinical implications of CRLF2 overexpression in Mexicans with ALL remain underexplored. Hence, this study aimed to investigate the prevalence of CRLF2 overexpression in Mexican patients with ALL across different age groups. Patients and Methods: We conducted a prospective study on a cohort of ALL patients from a single institution in Mexico that cares for patients across the age spectrum. Consecutive patients with a diagnosis of ALL were enrolled in the study and were assessed for CRLF2 overexpression using flow cytometry and fluorescent in situ hybridization (FISH). Patients were treated with a local BFM-based chemotherapy regimen according to age and risk stratification. In adolescents and young adults ≥16, a pediatric-inspired regimen was used and in patients with positive measurable residual disease (MRD) allogeneic hematopoietic cell transplantation was sought. To determine the impact of CRLF2 overexpression on clinical characteristics and treatment response, we analyzed various parameters, including response rates, MRD, and survival. Statistical analyses were performed using SPSS 25, encompassing descriptive analysis, chi-squared tests, and survival analysis with the Kaplan-Meier method. Results: A total of 79 patients were enrolled with a median age of 17 years (2-70 years), 34 were female and 45 males with a median white blood cell count of 10.1x103/L (.63-649.57). Our findings revealed that 21 out of 79 patients (26.6%) exhibited CRLF2 overexpression. The proportion of CRLF positive ALL increased with age; it was 20.5% in patients aged 0-16 years, 34.6% in 17-39 and 40% in >60 years. There were no statistically significant differences observed in white blood cell counts, central nervous system involvement, response to treatment, or minimal residual disease levels in CRLF2 positive or negative patients (Table 1). In the Kaplan-Meier survival analysis, it was observed that positive CRLF2 expression was significantly associated with lower overall survival (OS) with a median of 24.9 months (0.2-65.4 months); with 85.8% OS at 2 years in CRLF2 negative vs 41.2% in CRLF2 positive patients (Figure 1). Event-free survival rate at 2 years was also lower with 70.8% vs 35.3% at 2 years in CLRF2 positive patients. Moreover, this association was found to be consistent across all age subgroups, including patients aged 0-16, 17-39, and over 40 years (P < 0.05). Conclusion: Our study highlights the significance of CRLF2 overexpression as a cost-effective biomarker indicative of an unfavorable prognosis in ALL across the age spectrum. Early detection of this alteration through flow cytometry plays a vital role in risk stratification and should prompt the implementation of tailored therapeutic interventions to improve clinical outcomes. Additionally, our study underscores the importance of conducting population-specific research to gain comprehensive insights into the genetic landscape and disease characteristics of ALL in different ethnic groups.

Poor outcome of pediatric B-cell acute lymphoblastic leukemia associated with high level of CRLF2 gene expression in distinct molecular subtypes.

Overexpression of the cytokine receptor-like factor 2 (CRLF2) gene is the most common feature in the Philadelphia chromosome (Ph)-like subtype of B-cell acute lymphoblastic leukemia (B-ALL). However, the predictive value of CRLF2 overexpression for the prognosis of pediatric B-ALL patients remain controversial. The molecular mechanisms that upregulate CRLF2 expression level in patients has not been fully elucidated. In this study, the prognostic impact of CRLF2 expression level on molecular types of B-ALL in pediatric patients from Zhujiang Hospital (n = 111) was retrospectively analyzed. Youden index analysis was used to categorize CRLF2 expression into 3 groups, and these categories more precisely described the differences in the prognosis of patients with varying expression levels of CRLF2 in both the Zhujiang Hospital cohort and the TARGET cohort. We used the Zhujiang Hospital cohort as a discovery cohort to determine the cutoff value of CRLF2 expression. CRLF2-high patients accounted for approximately 6%. In addition, the percentage of bone marrow blast cells and initial white blood cell count in CRLF2-high patients were higher than those in CRLF2-low patients, and MRD turned negative slower. The results were validated in the TARGET cohort and indicated that CRLF2 overexpression could be subdivided by CRLF2 expression levels into 2 categories: CRLF2-high with a poor survival and CRLF2-medium with a good OS and EFS. Such heterogeneity was attributed to the different molecular mechanisms leading to CLRF2 upregulation, where the CRLF2 overexpression level was high in Ph-like B-ALL and medium in high hyperdiploid B-ALL. This study highlights the importance of the molecular mechanisms of the upregulation of CRLF2 expression in predicting the prognosis of pediatric B-ALL patients.

Long non-coding RNA MIR22HG suppresses the chondrogenic differentiation of human adipose-derived stem cells by interacting with CTCF to upregulate CRLF1.

Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. LncRNA MIR22HG has been proven to accelerate osteogenic differentiation, but the regulation mechanism of chondrogenic differentiation is still unclear. Human adipose-derived stem cells (hADSCs) have been widely utilised for bone tissue engineering applications. The present study aimed to examine the effect of MIR22HG on the chondrogenic differentiation of hADSCs. The results confirmed that MIR22HG was downregulated in the process of chondrogenic differentiation. Subsequently, gain- and loss-of-function of MIR22HG experiments showed that the overexpression of MIR22HG suppressed the deposition of cartilage matrix proteoglycans and decreased the expression of cartilage-related markers (e.g. Sox9, ACAN and Col2A1), whereas the knockdown of MIR22HG had the opposite effect. MIR22HG could bind to CTCF (CCCTC-binding factor), and CTCF could bind to the CRLF1 (cytokine receptor-like factor 1) promoter and upregulate CRLF1 gene expression. Besides, inhibition of CRLF1 can reverse the effect of MIR22HG on cell chondrogenic differentiation of hADSCs. Taken together, our outcomes reveal that MIR22HG suppressed chondrogenic differentiation by interaction with CTCF to stabilise CRLF1.

An antibody fragment-decorated liposomal conjugate targets Philadelphia-like acute lymphoblastic leukemia

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is an aggressive B-ALL malignancy associated with high rates of relapse and inferior survival rate. While targeted treatments against the cell surface proteins CD22 or CD19 have been transformative in the treatment of refractory B-ALL, patients may relapse due to antigen loss, necessitating targeting alternative antigens. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in half of Ph-like ALL cases conferring chemoresistance and enhancement of leukemia cell survival. Therefore, targeting CRLF2 may reduce the likelihood of relapse associated with antigen loss. We developed a CRLF2-targeting single-chain variable fragment modified by the fragment crystallizable region (CRLF2 scFv-Fc) conjugated to a drug maytansinoid 1 (DM1)-DOPC liposomal conjugate, creating homogeneous CRLF2-targeted liposomes (CRLF2-DM1 LIP). Cellular association and internalization studies in a Ph-like ALL cell line, MHH-CALL-4, compared to its lentivirally transduced CRLF2-knockdown counterpart (KD-CALL-4) revealed excellent CRLF2-targeting efficiency of CRLF2-DM1 LIP. Moreover, CRLF2-DM1 LIP showed selective association and internalization ex vivo using Ph-like ALL patient-derived xenograft (PDX) cells with minimal reactivity with non-target cells. Cell apoptosis assays demonstrated the CRLF2-dependent potency of CRLF2-DM1 LIP in Ph-like ALL cell lines. This study is the first to highlight the therapeutic potential of a CRLF2-directed scFv-Fc-liposomal conjugate for targeting Ph-like ALL.

Effect of N-glycosylation on constitutive signal transduction by mutated cytokine receptor-like factor 2

BackgroundCytokine receptor-like factor 2 (CRLF2) is a subunit of the receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation (insEIM) in the transmembrane domains of CRLF2 has been identified in acute lymphocytic leukemia (ALL), and Glu-Ile-Met (EIM) CRLF2 induces constitutive activation of signals. However, the signaling mechanism remains unclear. MethodsHEK293 cells were transfected with expression vectors encoding wild-type (WT), insEIM CRLF2, or their mutants which N-glycosylation site was replaced with a glutamine. Cell surface expression of CRLF2 was assessed by flow cytometry. Total CRLF2 and phosphorylated signal transducer and activator of transcription 5 (STAT5) were detected by western blotting. ResultsThree major species of CRLF2 (53-, 57- and 58-kDa) were identified. Deglycosylation analysis revealed that they were modified with complex-type and oligomannose-type glycans. The expression of both WT and EIM CRLF2 decreased in N-acetylglucosaminyltransferase (GnT)-I (MGAT1) knockout (KO) cells and slightly decreased in α1,6-fucosyltransferase (Fut8) KO cells compared to that in the control cells. In GnT-I or Fut8 KO cells, WT CRLF2 did not induce ligand-independent activation. Both WT and EIM CRLF2 contained four N-glycosylation sites. N55 of CRLF2 was required for the cell surface expression and activation by EIM CRLF2. ConclusionsWe found that N-glycosylation of CRLF2 plays crucial roles for its cell surface expression and signaling. However, N-glycan processing in the Golgi apparatus does not seem to be essential for ligand-independent activation of EIM CRLF2. General significanceOur studies provide a crucial role of glycosylation in the cell surface expression of receptors.

Cytokine receptor-like factor 1 (CRLF1) promotes cardiac fibrosis via ERK1/2 signaling pathway.

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

Gain of chromosome 21 increases the propensity for P2RY8::CRLF2 acute lymphoblastic leukemia via increased HMGN1 expression.

Acute lymphoblastic leukemia (ALL) patients with a gain of chromosome 21, intrachromosomal amplification of chromosome 21 (iAMP21), or Down syndrome (DS), have increased expression of genes in the DS critical region (DSCR) of chromosome 21, including the high-mobility group nucleosome-binding protein 1, HMGN1. Children with DS are predisposed to develop hematologic malignancies, providing insight into the role of chromosome 21 in the development of leukemias. A 320-kb deletion in the pseudoautosomal region of the X/Y chromosome in leukemic cells, resulting in a gene fusion between the purinergic receptor and cytokine receptor-like factor-2 (P2Y Receptor Family Member 8 (P2RY8)::CRLF2), is a common feature in ~60% of DS-ALL and ~40% of iAMP21 patients, suggesting a link between chromosome 21 and P2RY8::CRLF2. In an Australian cohort of pediatric B-ALL patients with P2RY8::CRLF2 (n = 38), eight patients harbored gain of chromosome 21 (+21), and two patients had iAMP21, resulting in a significantly increased HMGN1 expression. An inducible CRISPR/Cas9 system was used to model P2RY8::CRLF2 and investigate its cooperation with HMGN1. This model was then used to validate HMGN1 as an influencing factor for P2RY8::CRLF2 development. Using Cas9 to cleave the DNA at the pseudoautosomal region without directed repair, cells expressing HMGN1 favored repair, resulting in P2RY8::CRLF2 generation, compared with cells without HMGN1. CRISPR/Cas9 P2RY8::CRLF2 cells expressing HMGN1 exhibit increased proliferation, thymic stromal lymphopoietin receptor (TSLPR) expression, and JAK/STAT signaling, consistent with cells from patients with P2RY8::CRLF2. Our patient expression data and unique CRISPR/Cas9 modeling, when taken together, suggest that HMGN1 increases the propensity for P2RY8::CRLF2 development. This has important implications for patients with DS, +21, or iAMP21.

The cytokine receptor CRLF3 is a human neuroprotective EV-3 (Epo) receptor.

The evolutionary conserved orphan cytokine receptor-like factor 3 (CRLF3) has been implicated in human disease, vertebrate hematopoiesis and insect neuroprotection. While its specific functions are elusive, experimental evidence points toward a general role in cell homeostasis. Erythropoietin (Epo) is a major regulator of vertebrate hematopoiesis and a general cytoprotective cytokine. Erythropoietic functions mediated by classical Epo receptor are understood in great detail whereas Epo-mediated cytoprotective mechanisms are more complex due to involvement of additional Epo receptors and a non-erythropoietic splice variant with selectivity for certain receptors. In the present study, we show that the human CRLF3 mediates neuroprotection upon activation with the natural Epo splice variant EV-3. We generated CRLF3 knock-out iPSC lines and differentiated them toward the neuronal lineage. While apoptotic death of rotenone-challenged wild type iPSC-derived neurons was prevented by EV-3, EV-3-mediated neuroprotection was absent in CRLF3 knock-out neurons. Rotenone-induced apoptosis and EV-3-mediated neuroprotection were associated with differential expression of pro-and anti-apoptotic genes. Our data characterize human CRLF3 as a receptor involved in Epo-mediated neuroprotection and identify CRLF3 as the first known receptor for EV-3.

Clcf1/Crlf1a-mediated signaling is neuroprotective and required for Müller glia proliferation in the light-damaged zebrafish retina.

Zebrafish possess the innate ability to fully regenerate any neurons lost following a retinal injury. This response is mediated by Müller glia that reprogram and divide asymmetrically to produce neuronal precursor cells that differentiate into the lost neurons. However, little is understood about the early signals that induce this response. Ciliary neurotrophic factor (CNTF) was previously shown to be both neuroprotective and pro-proliferative within the zebrafish retina, however CNTF is not expressed following injury. Here we demonstrate that alternative ligands of the Ciliary neurotrophic factor receptor (CNTFR), such as Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine receptor-like factor 1a (Crlf1a), are expressed within Müller glia of the light-damaged retina. We found that CNTFR, Clcf1, and Crlf1a are required for Müller glia proliferation in the light-damaged retina. Furthermore, intravitreal injection of CLCF1/CRLF1 protected against rod photoreceptor cell death in the light-damaged retina and induced proliferation of rod precursor cells in the undamaged retina, but not Müller glia. While rod precursor cell proliferation was previously shown to be Insulin-like growth factor 1 receptor (IGF-1R)-dependent, co-injection of IGF-1 with CLCF1/CRLF1 failed to induce further proliferation of either Müller glia or rod precursor cells. Together, these findings demonstrate that CNTFR ligands have a neuroprotective effect and are required for induction of Müller glia proliferation in the light-damaged zebrafish retina.

The prognostic significance of CRLF2 expression at diagnosis in adult Ph-negative B-cell precursor acute lymphoblastic leukemia

The prognostic significance of cytokine receptor like factor 2 (CRLF2) expression at diagnosis in adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) needs to be clarified. A total of 357 bone marrow samples collected from consecutive adult cases with Ph-negative BCP-ALL at diagnosis retrospectively detected CRLF2 transcript levels by real-time quantitative PCR. Twenty percent was selected as the cutoff value for CRLF2 to divide patients into CRLF2_H and CRLF2_L groups. CRLF2_H was associated with higher WBC count, P2RY8–CRLF2 fusion and IKZF1 deletions (IKZF1del). In both the whole cohort and B-other patients, CRLF2_H independently predicted lower CR rates after induction. Furthermore, CRLF2_H/IKZF1del(+) patients had significantly lower CR, RFS, and OS rates and tended to have lower RFS and OS rates than others in the whole cohort and B-other patients, respectively. Therefore, coexistence of CRLF2_H and IKZF1del at diagnosis predicts poor response and outcome in adult Ph-negative BCP-ALL.

TSLP as a Potential Therapy in the Treatment of CRLF2 B Cell Acute Lymphoblastic Leukemia.

Cytokine receptor-like factor 2 B-cell acute lymphoblastic leukemia (CRLF2 B-ALL) is a high-risk subtype characterized by CRLF2 overexpression with poor survival rates in children and adults. CRLF2 and interleukin-7 receptor alpha (IL-7Rα) form a receptor for the cytokine thymic stromal lymphopoietin (TSLP), which induces JAK/STAT and PI3K/AKT/mTOR pathway signals. Previous studies from our group showed that low TSLP doses increased STAT5, AKT, and S6 phosphorylation and contributed to CRLF2 B-ALL cell survival. Here we investigated the role of TSLP in the survival and proliferation of CRLF2 B-ALL cells in vitro and in vivo. We hypothesized that high doses of TSLP increase CRLF2 signals and contribute to increased proliferation of CRLF2 B-ALL cells in vitro and in vivo. Interestingly, we observed the opposite effect. Specifically, high doses of TSLP induced apoptosis in human CRLF2 B-ALL cell lines in vitro, prevented engraftment of CRLF2 B-ALL cells, and prolonged the survival of +TSLP patient-derived-xenograft mice. Mechanistically, we showed that high doses of TSLP induced loss of its receptor and loss of CRLF2 signals in vitro. These results suggest that high doses of TSLP could be further investigated as a potential therapy for the treatment of CRLF2 B-ALL.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia

This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p=0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

High occurrence of CRLF2 abnormalities in Mexican children with B-cell acute lymphoblastic leukemia.

The P2RY8-CRLF2 and IGH-CRLF2 rearrangements induce the overexpression of cytokine receptor-like factor 2 (CRLF2) and have been associated with relapse and poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Additionally, they are frequently documented in high-risk Hispanic populations. To better understand the potential causes of the adverse prognosis of childhood B-ALL in Mexico, we analyzed these rearrangements and the CRLF2 mRNA and protein levels in 133 Mexican children with B-ALL. We collected bone marrow samples at diagnosis and evaluated the CRLF2 gene expression by qRT-PCR and the total CRLF2 protein by flow cytometry. P2RY8-CRLF2 and IGH-CRLF2 were detected by RT-PCR and FISH, respectively. The median time of follow-up to determine the prognostic significance of the CRLF2 abnormalities was three years. In 82% of the participants, the mRNA levels correlated with the cell-surface and intracellular CRLF2 protein levels. The P2RY8-CRLF2 rearrangement was present in 31.5% (42/133) of the patients, while the IGH-CRLF2 rearrangement was detected in 13.5% (9/67) of patients with high expression of CRLF2 (6.8% of the total sample). CRLF2 copy number variations (gain) were also detected in 7.5% (5/67) of patients with high protein levels. The overall survival (OS) presented significantly lower rates in patients with high white blood cell count (≥50x109/L) regardless of CRLF2 expression, but high levels of CRLF2 gene expression appears to contribute to the reduction of OS within this group of patients. In conclusion, in our cohort, a high occurrence of CRLF2 abnormalities was documented, particularly the P2RY8-CRLF2 rearrangement, which might represent a characteristic of the Mexican population. Targeted therapy to treat this group of patients could improve OS.

P1421: BIMODAL TARGETING OF CYTOKINE RECEPTOR-LIKE FACTOR 2 (CRLF2) WITH JAK INHIBITION AND CHIMERIC ANTIGEN RECEPTOR T CELL IMMUNOTHERAPY IN DOWN SYNDROME ACUTE LYMPHOBLASTIC LEUKEMIA

Background:CRLF2 (cytokine receptor-like factor 2; encoding the thymic stromal lymphopoietin receptor [TSLPR]) rearrangements occur in 50% of children with Down Syndrome-associated B-acute lymphoblastic leukemia (DS-ALL). These genetic alterations induce constitutive JAK/STAT and other kinase signaling that may be targetable by the JAK1/2 inhibitor ruxolitinib. Patients with DS-ALL experience substantial toxicity with conventional chemotherapy and particularly with hematopoietic stem cell transplantation (HSCT). The Children’s Oncology Group AALL1521 clinical trial (NCT02723994) is investigating the potential superior efficacy of ruxolitinib addition to chemotherapy for pediatric patients with CRLF2-rearranged/JAK pathway-mutant BCR-ABL1-like ALL, but children with DS-ALL are not eligible to participate in this study Successful development of precision medicine approaches that can increase cure, reduce treatment toxicity, and avoid HSCT remain a priority for children with D-ALL. Aims: We hypothesized that dual CRLF2 targeting with ruxolitinib and cellular immunotherapy could improve anti-leukemia activity against CRLF2-rearranged (R) DS-ALL. Methods: We previously reported preclinical activity of TSLPR-redirected chimeric antigen T cells with 4-1BB/CD3z costimulatory domains [TSLPRCART] against CRLF2-overexpressing ALL cells (Qin Blood 2015). In the present studies, we investigated the effects of TSLPRCART +/- ruxolitinib co-treatment in vitro and in vivo against CRLF2-R human ALL cell lines and childhood DS-ALL patient-derived xenograft (PDX) models (n=4). Results:In vitro incubation of CD28/CD3 bead-activated normal human T cells or TSLPRCART with ruxolitinib significantly diminished IL-2 and IFN-ɣ cytokine production, although did not induce overt apoptosis of normal or engineered T cells. As expected, treatment of CRLF2-R DS-ALL PDX models with TSLPRCART potently inhibited in vivo leukemia proliferation and induced long-term ‘cure’ of animals. However, simultaneous co-treatment of engrafted mice with ruxolitinib and TSLPRCART led to markedly diminished in vivo T cell proliferation and persistence, blunted cytokine production, and facilitated eventual leukemia relapse (representative data in Figure 1). These deleterious effects could be abrogated by 14-day delayed sequencing of ruxolitinib after TSLPRCART activation and expansion and were further fully reversible after ruxolitinib withdrawal, resulting in renewed TSLPCART functionality over time and upon leukemia rechallenge. Image:Summary/Conclusion: TSLPRCART is a promising cellular immunotherapeutic approach for DS-ALL and for BCR-ABL1-like ALL given shared features of CRLF2 rearrangement and cell surface protein overexpression. In the present studies, we demonstrate that ruxolitinib co-administration in close proximity impaired in vivo TSLPRCART-induced DS-ALL cell killing but was also beneficial in protection against life-threatening hyperinflammation/cytokine release syndrome (CRS) in dual-treated animals. These results suggest potential for both anti-ALL efficacy and CRS dampening when JAK inhibition and TSLPRCART are properly time-sequenced and may have broader applicability for toxicity mitigation with other cellular immunotherapies. Studies of CAR T cell immunotherapy and other kinase inhibitors in additional CRLF2-rearranged and non-rearranged DS-ALL models are ongoing.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.

Abstract 1528: Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of leukemia in children. B-ALL characterized by cytokine receptor-like factor 2 (CRLF2) overexpression (CRLF2 B-ALL) has a survival rate of &amp;lt;30% and is the highest risk sub-group of B-ALL in both adults and children. CRLF2 is a receptor component for the cytokine thymic stromal lymphopoietin (TSLP). TSLP plays a role in the survival and proliferation of B-cell precursors, thus explaining the oncogenic role of increased CRLF2 signaling in CRLF2 B-ALL. To our surprise, we found that high-levels of TSLP eliminated leukemia cells in patient-derived xenograft (PDX) models of CRLF2 B-ALL. CRLF2 and IL-7 receptor-alpha (IL-7Ra) form the heterodimer type-I cytokine receptor for TSLP cytokine. Binding of TSLP to its CRLF2 receptor complex induces JAK-STAT5 and PI3K-AKT pathway signals. TSLP shares the IL-7Ra with Interleukin 7 (IL-7) which has a heterodimer receptor consisting of IL-7Ra and the common gamma chain. High-levels of IL-7 (50 ng/ml) have been shown to induce IL-7Ra internalization and degradation in T-cells. We hypothesize that high-level TSLP induces internalization and degradation of IL-7Ra leading to CRLF2 signal inhibition, death of CRLF2 B-ALL cells and the anti-leukemia effects that we have observed in PDX mice. To test this hypothesis, we treated CRLF2 B-ALL cell lines with different TSLP concentrations to observe the effect of TSLP on its receptor and CRLF2 signaling. Flow cytometry data showed that continuous or a pulse of high-dose TSLP induced a loss of surface IL-7Ra expression for up to 24 hours. Phosphorylation assays showed that cells cultured with high-dose TSLP were unresponsive to subsequent TSLP-induced phosphorylation events (pSTAT5 and pRPS6), indicating CRLF2 signal inhibition. In conclusion, high-dose TSLP induces loss of (IL-7Ra) and inhibition of CRLF2 signaling. These results suggest that TSLP exerts its anti-leukemia effects by shutting down CRLF2-mediated signals possibly via the loss of the IL-7Ra receptor component. Citation Format: Hossam Alkashgari, Cornelia Stoian, Caleb Ruiz-Jimenez, Jacqueline Coats, Carlos A. Casiano, Sinisa Dovat, Kimberly J. Payne. Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1528.