JAK Inhibitor Ruxolitinib Research Articles

Prevention of liver metastasis via the pharmacological suppression of AMIGO2 expression in tumor cells.

AMIGO2 adheres to liver endothelium and induces liver metastasis. We revealed that genetically altering AMIGO2 expression in tumor cells affects their liver metastatic potential, depending on the AMIGO2 expression level. The aim of this study was to prevent liver metastasis by pharmacologically suppressing AMIGO2 expression. For screening, we used the mouse LV12 cells because of their affinity to adhere to liver endothelium and metastasize the liver owing to elevated AMIGO2 expression. Of the 285 compounds tested, 17 reduced AMIGO2 mRNA expression. We subsequently screened for compounds that inhibited tumor cell adhesion to liver endothelium and identified five compounds that inhibit three signaling pathways (MEK, JAK, and JNK). Treatment with these compounds inhibited liver metastasis of LV12 cells. Next, we used clinically available signal inhibitors (MEK inhibitor trametinib, JAK inhibitor ruxolitinib, and JNK inhibitor SP600125), and found that ruxolitinib inhibits AMIGO2 expression more stably. Furthermore, ruxolitinib inhibited the adhesion of LV12 cells to liver endothelium and suppressed liver metastasis. Using the MKN45 gastric cancer cells, we confirmed that ruxolitinib could prevent liver metastasis of human cancer cells. These results demonstrate that pharmacological inhibition of AMIGO2 expression in tumor cells is a promising novel strategy to prevent and control liver metastasis.

EPCO-43. DECONVOLUTION OF GLIOBLASTOMA RNA-SEQ REVEALS IMMUNE SUBPOPULATIONS PREDICTING THERAPEUTIC RESPONSE

Abstract Bulk RNA-seq is now among the standard of care for patients with newly diagnosed glioblastoma (GBM), but whether this data can predict therapeutic response is unclear. In silico deconvolution of bulk RNA-seq data allows for quantification of cell types comprising the tumor and its microenvironment. Our study tests whether cell type proportions predict therapeutic response in patients with GBM and high-grade glioma (HGG) enrolled in trials of investigational agents. 127 patients with GBM/HGG underwent post-resection/biopsy whole exome RNA-seq and reads were post-processed using standard computational pipelines. In silico deconvolution was performed using GBMDeconvoluteR, which generates cellular proportions comprising the RNA-seq reads. Multivariate Cox regression models were used to examine each cell proportion as a predictor of progression-free (PFS) and overall (OS) survival. Statistical significance was ascertained using Bonferroni correction for multiple comparisons. Among 54 patients treated with a PD-L1 inhibitor (nivolumab) and VEGF inhibitor (bevacizumab), the proportions of NK cells (p=0.0024), radial glial cells (p=0.0010), and mural cells (p=0.0025), was associated with increased PFS, while proportion of endothelial cells (p=0.0008) was associated with decreased PFS. Astrocyte (p=0.0022), macrophage (p=0.0015), NK cell (p<0.0001), and mural cell (p=0.0004) proportions were associated with longer OS, while endothelial cell proportion (p=0.0001) was associated with shorter OS. Among 21 patients treated with a tyrosine kinase inhibitor (ibrutinib) combined with radiation and temozolomide (RT/TMZ), cell proportions assessed at the time of diagnosis did not predict PFS or OS. In 52 patients with HGG treated with a JAK inhibitor (ruxolitinib) with RT/TMZ, only mural cell proportion was associated with a longer OS (p=0.0017). Our results highlight how bulk RNA-seq data can be used to predict therapeutic response. In GBM patients treated with immunotherapies, the proportion of NK cells may predict response. Further work will examine which gene programs are predictive of therapeutic response.

Reprogramming cellular senescence in the tumor microenvironment augments cancer immunotherapy through multifunctional nanocrystals.

Harnessing the immunogenic potential of senescent tumor cells provides an opportunity to remodel tumor microenvironment (TME) and boost antitumor immunity. However, this potential needs to be sophisticatedly wielded to avoid additional immunosuppressive capacity of senescent cells. Our study shows that blocking the JAK2/STAT3 pathway enhances immunogenic efficacy of Aurora kinase inhibitor alisertib (Ali)-induced senescence by reducing immunosuppressive senescence-associated secretory phenotype (SASP) while preserving immunogenic SASP. Hypothesizing that SASP reprogramming with Ali and JAK2 inhibitor ruxolitinib (Rux) will benefit cancer immunotherapy, we create nanoparticulate crystals (Ali-Rux) composed of Ali and Rux with a fully active pharmaceutical ingredient. Immunization with Ali-Rux-orchestrated senescent cells promotes stronger activation of antigen-presenting cells, enhancing antitumor immune surveillance. This approach remodels the TME by increasing CD8+ T cell and NK recruitment and activation while decreasing MDSCs. Combined with PD-L1 blockade, Ali-Rux elicits a durable antitumor immune response, suggesting the TME reshaping approach as a potential cancer immunotherapy.

SO2 activates Th17 cells through the JAK1,2/STAT3 signaling pathway

ObjectiveTo investigate the effect of SO2 on Th1/Th2/Th17 cells in allergic rhinitis (AR) and the role of JAK1, 2/STAT3 signaling pathways.To Provide potential directions for the treatment of AR. MethodsFifteen AR patients were enrolled as the experimental group, while 15 healthy volunteers served as the normal control group. After collecting venous blood, peripheral blood mononuclear cells (PBMCs) were isolated and cultured, followed by the addition of SO2 derivatives and the JAK inhibitor Ruxolitinib. Flow cytometry was employed to assess alterations in the Th1/Th2 and Th17/Treg cell balance upon stimulation with SO2 and Ruxolitinib. qRT-PCR was utilized to detect the expression of Th1-related cytokines IL-2 and IFN-γ, Th2-related cytokines IL-4 and IL-5, Th17-related cytokines IL-17A and RORγt, as well as genes JAK1, JAK2, and STAT3. Flow cytometric cytokine analysis was conducted for quantitative assessment of the expression levels of inflammation-related cytokines in PBMC culture supernatants after stimulation. In addition, we stimulated the Jurkat T lymphocyte cell line with SO2 derivatives, added Ruxolitinib as an inhibitor, and used Western blot analysis to further determine the effects of SO2 on Th cells and the role of the JAK1,2/STAT3 signaling pathway in this process. ResultsStimulation with SO2 derivatives upregulated the expression levels of Th2 cells and associated cytokines, as well as Th1 cells and associated cytokines. both AR patients and healthy individuals displayed increased percentages of Th17 cells and Th17/Treg ratios in PBMCs. The expression of IL-17A, RORγt, and IL-6 was also elevated. Under SO2 stimulation, the expression of JAK1, JAK2, STAT3, and RORγt in Jurkat cells increased. Moreover, after the application of Ruxolitinib, the JAK/STAT signaling pathway was inhibited. This led to a reduction in Th17 cells and IL-17A levels in both AR patients and healthy individuals, as well as a decrease in RORγt expression in Jurkat cells. Additionally, the expression of IL-5 decreased in healthy individuals. ConclusionSO2 exposure exacerbated Th1/Th2/Th17 inflammation in AR patients and induced Th1 and Th17 inflammation in healthy individuals. The stimulatory effect of SO2 on Th17 cell differentiation could be inhibited by Ruxolitinib. This suggests that the Th17 inflammation induced by SO2 stimulation may be related to the activation of the JAK/STAT signaling pathway, and this has been confirmed in the Jurkat cell line.

High extracellular matrix stiffness promotes the metastasis of nasopharyngeal carcinoma through STAT3/FGF1 positive feedback regulation activated by JAK2

The stiffness of the extracellular matrix (EM) is known to be associated with tumor metastasis. This study aims to clarify the function and mechanisms of EM stiffness in nasopharyngeal carcinoma (NPC) and to provide a potential therapeutic target for patients with NPC. The NPC cell lines were cultured under varying stiffnesses. Plate cloning, Transwell assays and scratch tests were conducted to compare the phenotypes. Transcriptome sequencing and enrichment analysis were performed to explore the key molecules and the downstream signaling pathways, which were verified via in vitro and in vivo experiments. Our study showed that the NPC cells cultured on a stiff substrate (50 ​kPa) had greater colony formation, invasion and migration abilities than those cultured on a soft substrate. Transcriptome sequencing showed that a stiff environment induced high expression of fibroblast growth factor 1 (FGF1), which suggested a poor prognosis in various malignancies. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway played an important role in NPC metastasis activated by the FGF1 autocrine pathway in a stiff environment. The increased FGF1 expression was regulated by the transcription factor STAT3, and a positive feedback regulation between them was observed. The use of the JAK inhibitor Ruxolitinib and JAK2 interference inhibited the activation of STAT3. In conclusion, JAK2 activates STAT3/FGF1 positive feedback regulation and promotes NPC metastasis via the PI3K/Akt signaling pathway in a high-stiffness environment. Hence, FGF1 can be used as a potential therapeutic target for patients with NPC.

Preventing periprosthetic osteolysis in aging populations through lymphatic activation and stem cell-associated secretory phenotype inhibition

With increases in life expectancy, the number of patients requiring joint replacement therapy and experiencing periprosthetic osteolysis, the most common complication leading to implant failure, is growing or underestimated. In this study, we found that osteolysis progression and osteoclast differentiation in the surface of the skull bone of adult mice were accompanied by significant expansion of lymphatic vessels within bones. Using recombinant VEGF-C protein to activate VEGFR3 and promote proliferation of lymphatic vessels in bone, we counteracted excessive differentiation of osteoclasts and osteolysis caused by titanium alloy particles or inflammatory cytokines LPS/TNF-α. However, this effect was not observed in aged mice because adipogenically differentiated mesenchymal stem cells (MSCs) inhibited the response of lymphatic endothelial cells to agonist proteins. The addition of the JAK inhibitor ruxolitinib restored the response of lymphatic vessels to external stimuli in aged mice to protect against osteolysis progression. These findings suggest that inhibiting SASP secretion by adipogenically differentiated MSCs while activating lymphatic vessels in bone offers a new method to prevent periprosthetic osteolysis during joint replacement follow-up.

TYK2 as a novel therapeutic target in Alzheimer's Disease with TDP-43 inclusions.

Neuroinflammation is a pathological feature of many neurodegenerative diseases, including Alzheimer's disease (AD)1,2 and amyotrophic lateral sclerosis (ALS)3, raising the possibility of common therapeutic targets. We previously established that cytoplasmic double-stranded RNA (cdsRNA) is spatially coincident with cytoplasmic pTDP-43 inclusions in neurons of patients with C9ORF72-mediated ALS4. CdsRNA triggers a type-I interferon (IFN-I)-based innate immune response in human neural cells, resulting in their death4. Here, we report that cdsRNA is also spatially coincident with pTDP-43 cytoplasmic inclusions in brain cells of patients with AD pathology and that type-I interferon response genes are significantly upregulated in brain regions affected by AD. We updated our machine-learning pipeline DRIAD-SP (Drug Repurposing In Alzheimer's Disease with Systems Pharmacology) to incorporate cryptic exon (CE) detection as a proxy of pTDP-43 inclusions and demonstrated that the FDA-approved JAK inhibitors baricitinib and ruxolitinib that block interferon signaling show a protective signal only in cortical brain regions expressing multiple CEs. Furthermore, the JAK family member TYK2 was a top hit in a CRISPR screen of cdsRNA-mediated death in differentiated human neural cells. The selective TYK2 inhibitor deucravacitinib, an FDA-approved drug for psoriasis, rescued toxicity elicited by cdsRNA. Finally, we identified CCL2, CXCL10, and IL-6 as candidate predictive biomarkers for cdsRNA-related neurodegenerative diseases. Together, we find parallel neuroinflammatory mechanisms between TDP-43 associated-AD and ALS and nominate TYK2 as a possible disease-modifying target of these incurable neurodegenerative diseases.

A phase Ib, open-label study of add-on therapy with CK0804 in participants with myelofibrosis and suboptimal response to ruxolitinib.

6580 Background: Suboptimal response to JAK2 inhibitor ruxolitinib in patients with myelofibrosis (MF) remains a major unmet medical need. Deregulated inflammatory pathways including CXCR4/CXCR12 axis might limit the therapeutic efficacy of ruxolitinib and contribute to disease progression. In preclinical studies, cord blood-derived, CXCR4 enriched T regulatory cells [CK0804; T-regs] showed ability to suppress inflammatory cytokines playing major role in MF (Huang et al., Cytotherapy, 2023). Methods: This phase Ib study evaluates the safety and activity of up to 6 doses of CK0804 (non-HLA matched, Cryopreserved) T-regs therapy, fixed dose of 100 million cells, added every 28 days to ruxolitinib. Patients with MF on ruxolitinib for ≥12 weeks and stable dose for ≥8 weeks, who have palpable splenomegaly, symptoms, or grade 2 cytopenia are eligible. Primary objective is safety, secondary objective is overall response per IWG-MRT criteria at 24 weeks. Present analysis includes cohort of initial run-in safety phase. Results: Nine patients of median age 68 years (range, 55-84) were enrolled, 44% were males. Median blood counts [range] showed while blood cells 10.6 [3.1-70.4] x10^9/L, hemoglobin 8.8 [7.8-11.5] g/dL, platelets 177 [148-311] x10^9/L. Three patients were transfusion dependent. Median spleen volume was 1449 cubic centimeters (176-5609), 7 patients (78%) had clinically worsening splenomegaly. Median symptoms score (MPN-SAF TSS) was 23 (20-40). Six (67%) had diploid karyotype, 5 (56%) had JAK2 mutation and 8 (89%) had additional no-driver mutations. Median duration of prior ruxolitinib was 35 months (range, 10-132), all patients were on ≥ 10 mg twice daily dose with no dose change throughout the study. Four patients received all six doses of CK0804, three patients have ongoing treatments. One patient experienced infusion reaction to second dose of CK0804 likely due to the excipient dimethylsulfoxide and withdrew consent. One patient died of unrelated cause prior to the infusion 6. There were no nonhematologic or hematologic adverse events. Two transfusion dependent patients who were evaluable for response had decreased monthly need for transfusions by the end of the sixth cycle: 4 to 2.8 units and 1.2 to 0.8 units, respectively. All patients noticed symptoms improvement; median best decline in MPN-SAF TSS was -38% (range, -20% to -71%); with ≥ 50% reduction in 3 patients. One patient achieved ≥ 35% spleen volume reduction at week 12. Longitudinal analysis of markers of inflammation is in progress and will be presented at the conference. Conclusions: This preliminary analysis of run-in phase of study evaluating CXCR4 enriched T regs cell therapy as addition to ruxolitinib shows initial safety with no myelosuppressive adverse events and promising clinical activity. Active enrollment of participants to the expansion cohort is ongoing. Clinical trial information: NCT05423691 .

#760 Monogenic lupus with ANCA associated vasculitis-phenotype and ANCA positivity due to DNASE1L3 mutations: case report and review of the literature

Abstract Background and Aims Monogenic systemic lupus erythematosus (SLE) accounts for 7-10% of cases of early-onset SLE. DNASE1L3 is an extracellular nuclease that digests chromatin released from apoptotic cells and its deficiency, enhance autoantibody production and type I interferon responses and cause different autosomal recessive phenotypes including SLE and hypocomplementemic urticarial vasculitis syndrome (HUVS) [1]. In some cases patients can have a mixed SLE-AAV phenotype. Kidney involvement in patients with DNASE1L3 mutations is poorly characterised. Method We present the case report of a 7-years-old boy with monogenic SLE due to DNASE1L3 mutations who developed severe glomerulonephritis leading to kidney failure. Results The patient had a history of urticaria and arthralgia and presented with fever, haemolytic anaemia, lymphadenopathy, hepato-splenomegaly, erythematous- macular and necrotic skin lesions, and interstitial lung disease. He also had acute kidney injury (serum creatinine 5.4 mg/dL), nephrotic- range proteinuria (3.9 g/24 h) and microhaematuria. Immunological tests revealed low C3 and C4, positive anti-nuclear antibody (ANA) and myeloperoxidase-antineutrophil cytoplasmic antibodies (MPO-ANCA), with negativity of anti-extractable nuclear antigen antibodies (anti-ENA) and anti-double stranded DNA (anti-dsDNA). Kidney biopsy showed pauci-immune (mesangial C3+ and IgG+) necrotising crescentic glomerulonephritis. The patient proved refractory to standard immunosuppression with cyclophosphamide and steroids. Two years later, due to renal function decline, a new kidney biopsy was performed and showed diffuse necrotising glomerulonephritis with endo- and extra-capillary proliferation; intense mesangial and subendothelial full-house deposits were evident on immunofluorescence. The patient did not respond to other lines of immunosuppression (mycophenolate mofetil, tacrolimus, azathioprine, rituximab), including the JAK inhibitor ruxolitinib. Kidney failure progressed to end stage at the age of 15 years, when the patient began haemodialysis. The kidney presentation, immunological profile and the first kidney biopsy led to diagnosis of AAV. However, the diagnosis was questioned due to the refractory course, the systemic clinical presentation and the second kidney histology (consistent with lupus nephritis). High interferon signature was detected and in the suspicion of a monogenic lupus nephritis Whole Exome Sequencing was performed and revealed homozygous DNASE1L3 variants (c.290_291delCA p.T97Ifs*2); thus, a final diagnosis of DNASE1L3 monogenic SLE was made. DNASE1L3 serum levels were normal but the DNAse enzymatic activity was low. Conclusion Renal involvement is one of the most frequent manifestations of DNASE1L3-related lupus; in the literature 32 cases of DNASE1L3-associated lupus nephritis are described. ANCA are positive in about 55% of these cases and are associated with a mixed lupus-AAV phenotype. All cases are characterized by resistance to common immunosuppressors and poor renal prognosis [2]. DNASE1L3 regulates neutrophil extracellular trap (NET) clearance and when this function is reduced the permanence of NETs induces endothelial damage and autoantibody formation. This pathogenic pathway is shared both by SLE and by AAV. There is no association between specific mutations and antibody positivity or clinical manifestations.

Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib

IntroductionThe signal transducer and activator of transcription (STAT1) gain-of-function (GOF) syndrome accounts for most cases of chronic mucocutaneous candidiasis but is characterized by a broader clinical phenotype that may include bacterial, viral, or invasive fungal infections, autoimmunity, autoinflammatory manifestations, vascular complications, or malignancies. The severity of lymphopenia may vary and influence the infectious morbidity.MethodsIn our cohort of seven STAT1-GOF patients, we investigated the mechanisms that may determine T lymphopenia, we characterized the interferon gene signature (IGS) and analyzed the effect of ruxolitinib in reverting the immune dysregulation.ResultsSTAT1-GOF patients exhibited increased T lymphocyte apoptosis that was significantly augmented in both resting conditions and following stimulation with mitogens and IFNα, as evaluated by flow cytometry by Annexin V/ Propidium iodide assay. The JAK inhibitor ruxolitinib significantly reduced the IFNα-induced hyperphosphorylation of STAT1 and reverted the stimulation-induced T-cell apoptosis, in vitro. In two adult STAT1-GOF patients, the JAKinib treatment ameliorated chronic mucocutaneous candidiasis and lymphopenia. Most STAT1-GOF patients, particularly those who had autoimmunity, presented increased IGS that significantly decreased in the two patients during ruxolitinib treatment.ConclusionIn STAT1-GOF patients, T lymphocyte apoptosis is increased, and T lymphopenia may determine higher risk of severe infections. The JAKinib target therapy should be evaluated to treat severe chronic candidiasis and lymphopenia, and to downregulate the IFNs in patients with autoinflammatory or autoimmune manifestations.

Safety and Feasibility of JAK Inhibitor Ruxolitinib in Newly-Diagnosed High-Grade Gliomas (CRUX): Final Trial Report

INTRODUCTION: Dysregulation of the JAK/STAT pathway in newly-diagnosed high-grade gliomas (nHGG) is linked to enhanced survival and proliferation of tumor cells. Ruxolitinib, a small molecule inhibitor of JAK1, JAK2, and JAK3, limits glioma growth in preclinical models. METHODS: This non-randomized prospective study included 60 WHO Grade 3-4 nHGG patients who received standard of care (SOC) therapy along with ruxolitinib in a 3 + 3 dose-escalation design; level 1 of 10 mg BID, level 2 of 15 mg BID, level 3 of 20 mg BID, level -1 of 5 mg BID. Primary study objective was determination of maximum tolerated dose (MTD) of ruxolitinib in combination with chemoradiation. Secondary objective was the determination of safety as assessed using NIH CTCAE scale, overall survival (OS), and progression-free survival (PFS). Exploratory aims were to investigate relationships between clinical outcomes and genomic signatures. RESULTS: 60 patients were enrolled, with median age 60.5 years (range 22-78), 23 (38%) female, and 37 (62%) were male. Amongst 59 evaluable patients, 29 (49%) were MGMT unmethylated and received ruxolitinib with 60Gy radiation over 6 weeks, while 30 (51%) patients were MGMT methylated and received ruxolitinib with 75mg/m2 of temozolomide (TMZ) with 60Gy radiation over 6 weeks. MTD for both cohorts was 20 mg BID. No dose-limiting toxicities were observed. Toxicities attributable to study medications included four grade 4 AEs including seizure, respiratory distress, somnolence, and thromboembolic event along with 14 grade 3 adverse events (AEs), including respiratory distress, seizure, gait disturbance, weakness, thrombocytopenia, cognitive disturbance, urinary retention, and meningitis. Median follow-up of patients alive was 34.2 months (range 18-52.2) with outcomes. CONCLUSIONS: Ruxolitinib therapy is safe and therapeutically feasible in combination with TMZ and radiation. A randomized phase 2 trial is planned.

SHP2 inhibition displays efficacy as a monotherapy and in combination with JAK2 inhibition in preclinical models of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, are clonal hematopoietic neoplasms driven by mutationally activated signaling by the JAK2 tyrosine kinase. Although JAK2 inhibitors can improve MPN patients' quality of life, they do not induce complete remission as disease-driving cells persistently survive therapy. ERK activation has been highlighted as contributing to JAK2 inhibitor persistent cell survival. As ERK is a component of signaling by activated RAS proteins and by JAK2 activation, we sought to inhibit RAS activation to enhance responses to JAK2 inhibition in preclinical MPN models. We found the SHP2 inhibitor RMC-4550 significantly enhanced growth inhibition of MPN cell lines in combination with the JAK2 inhibitor ruxolitinib, effectively preventing ruxolitinib persistent growth, and the growth and viability of established ruxolitinib persistent cells remained sensitive to SHP2 inhibition. Both SHP2 and JAK2 inhibition diminished cellular RAS-GTP levels, and their concomitant inhibition enhanced ERK inactivation and increased apoptosis. Inhibition of SHP2 inhibited the neoplastic growth of MPN patient hematopoietic progenitor cells and exhibited synergy with ruxolitinib. RMC-4550 antagonized MPN phenotypes and increased survival of an MPN mouse model driven by MPL-W515L. The combination of RMC-4550 and ruxolitinib, which was safe and tolerated in healthy mice, further inhibited disease compared to ruxolitinib monotherapy, including extending survival. Given SHP2 inhibitors are undergoing clinical evaluation in patients with solid tumors, our preclinical findings suggest that SHP2 is a candidate therapeutic target with potential for rapid translation to clinical assessment to improve current targeted therapies for MPN patients.

Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

Incidence of blast phase in myelofibrosis patients according to anemia severity at ruxolitinib start and during therapy.

Anemia is frequently present in patients with myelofibrosis (MF), and it may be exacerbated by treatment with the JAK2-inhibitor ruxolitinib (RUX). Recently, a relevant blast phase (BP) incidence has been reported in anemic MF patients unexposed to RUX. The authors investigated the incidence of BP in 886 RUX-treated MF patients, included in the "RUX-MF" retrospective study. The BP incidence rate ratio (IRR) was 3.74 per 100 patient-years (3.74 %p-y). At therapy start, Common Terminology Criteria for Adverse Events grade 3-4 anemia (hemoglobin [Hb] <8g/dL) and severe sex/severity-adjusted anemia (Hb <8/<9g/dL in women/men) were present in 22.5% and 25% patients, respectively. IRR of BP was 2.34 in patients with no baseline anemia and reached respectively 4.22, 4.89, and 4.93 %p-y in patients with grade 1, 2, and 3-4 anemia. Considering the sex/severity-adjusted Hb thresholds, IRR of BP was 2.85, 4.97, and 4.89 %p-y in patients with mild/no anemia, moderate, and severe anemia. Transfusion-dependent patients had the highest IRR (5.03 %p-y). Progression-free survival at 5years was 70%, 52%, 43%, and 27% in patients with no, grade 1, 2, and 3-4 anemia, respectively (p<.001). At 6months, 260 of 289 patients with no baseline anemia were receiving ruxolitinib, and 9.2% had developed a grade 3-4 anemia. By 6-month landmark analysis, BP-free survival was significantly worse in patients acquiring grade 3-4 anemia (69.3% vs. 88.1% at 5years, p<.001). This study highlights that anemia correlates with an increased risk of evolution into BP, both when present at baseline and when acquired during RUX monotherapy. Innovative anemia therapies and disease-modifying agents are warranted in these patients.

ACVR1: A Novel Therapeutic Target to Treat Anemia in Myelofibrosis.

Activin receptor type I (ACVR1) is a transmembrane kinase receptor belonging to bone morphogenic protein receptors (BMPs). ACVR1 plays an important role in hematopoiesis and anemia via the BMP6/ACVR1/SMAD pathway, which regulates expression of hepcidin, the master regulator of iron homeostasis. Elevated hepcidin levels are inversely associated with plasma iron levels, and chronic hepcidin expression leads to iron-restricted anemia. Anemia is one of the hallmarks of myelofibrosis (MF), a bone marrow (BM) malignancy characterized by BM scarring resulting in impaired hematopoiesis, splenomegaly, and systemic symptoms. Anemia and red blood cell transfusions negatively impact MF prognosis. Among the approved JAK inhibitors (ruxolitinib, fedratinib, momelotinib, and pacritinib) for MF, momelotinib and pacritinib are preferably used in cytopenic patients; both agents are potent ACVR1 inhibitors that suppress hepcidin expression via the BMP6/ACVR1/SMAD pathway and restore iron homeostasis/erythropoiesis. In September 2023, momelotinib was approved as a treatment for patients with MF and anemia. Zilurgisertib (ACVR1 inhibitor) and DISC-0974 (anti-hemojuvelin monoclonal antibody) are evaluated in early phase clinical trials in patients with MF and anemia. Luspatercept (ACVR2B ligand trap) is assessed in transfusion-dependent MF patients in a registrational phase 3 trial. Approved ACVR1 inhibitors and novel agents in development are poised to improve the outcomes of anemic MF patients.

Abstract C099: Establishment and characterization of an NUP98-KDM5A-driven AML XPDX model

Abstract Background: Acute myeloid leukemia (AML) with NUP98 fusions is associated with poor prognosis in adults and especially children. Pediatric AML is a rare and heterogeneous disease characterized by a high prevalence of gene fusions as driver mutations, including the NUP98-KDM5A fusion found in a particular subgroup of patients with complex karyotypes. To better understand this subtype of AML, we established and characterized a XPDX model designated ST1653 representing NUP98-KDM5A-driven disease from a previously treated AML patient. We performed DNA and RNA-based sequencing of the model. Further we evaluated in vivo sensitivity towards cytarabine and inhibitors targeting the JAK and CDK6 pathways, which have been shown to be important in NUP98-KDM5A-driven AML. Methods: ST1653 was established from a bone marrow aspirate collected from a 63-year-old Caucasian male who had AML recurrence following high dose cytarabine. The sample was injected subcutaneously into a triple-deficient mouse and the P0 model passaged after seventy days into CB-17 SCID and athymic nude mice and further developed until stable. WES and RNAseq were then performed. For in vivo studies, ST1653 was evaluated against cytarabine and the JAK inhibitors ruxolitinib, baricitinib and tofacitinib and the CDK4/6 inhibitors palbociclib, abemaciclib and ribociclib. Endpoints in all studies included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0%) versus Day 0 tumor volume was also reported. Results: Growth of the ST1653 AML XPDX model was similar in CB-17 SCID or athymic nude mice with a median time to endpoint of forty-five days, although the take rate in the SCID strain was superior. Sequencing identified the presence of the NUP98-KDM5A fusion and chimeric protein expression was confirmed. In vivo, ST1653 was insensitive to cytarabine and the JAK inhibitors but demonstrated limited sensitivity to CDK4/6i treatment. Conclusion: We have established and characterized a XPDX model of NUP98-KDM5A-driven AML including sequencing and in vivo evaluation. This model is an important tool for developing novel therapies to this high-risk leukemia subtype. Citation Format: Armando Diaz III, Alyssa Simonson, Johnnie Flores, Joseph Holahan, Kyriakos P Papadopoulos, Jim Lund, Drew Rasco, Amita Patnaik, Michael J Wick. Establishment and characterization of an NUP98-KDM5A-driven AML XPDX model [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 C099.

TGFβ1 Secretion in Megakaryocytes Is Autophagy-Dependent and Its Inhibition Ameliorates Myelofibrosis in Mice

Megakaryocytes (MKs) are large, polyploid platelet precursor cells that primarily reside within the bone marrow. Recent advances revealed that in addition to platelet production, MKs contribute to immunity and maintenance of bone marrow homeostasis, raising the question of whether these functions are hijacked in disease. One hallmark of primary and secondary myelofibrosis (MF), subsets of myeloproliferative neoplasms, is the accumulation of aberrant, immature MKs in the bone marrow. These release pro-fibrotic cytokines such as transforming growth factor β1 (TGFβ1) and interleukin 1β (IL1β) into the bone marrow, thus inducing fibrotic alterations that ultimately cause pancytopenia - the major cause of mortality in patients with MF. While current treatment strategies in MF aim to prevent MK proliferation and correct the differentiation defects, a direct targeting of cytokine release from MKs has not yet been explored. Recently, unconventional secretion of TGFβ1 and IL1β via secretory autophagy, a secretion pathway driven by the fusion of autophagosomes with the plasma membrane, has been shown to occur in other cell types. We therefore hypothesize that MKs may preferentially utilize secretory autophagy for cytokine release and that inhibiting autophagosome formation might therefore slow fibrosis progression in vivo. Previous studies suggested that TGFβ1 is stored within specialized α-granules in MKs. To test this, we cultured murine MKs and determined TGFβ1 co-localization with different α-granule proteins by super-resolution confocal microscopy. However, while we did not identify any correlation between TGFβ1 and α-granules, we found that TGFβ1 strongly co-localized with the autophagy marker light chain (LC) 3B, supporting a potential role for secretory autophagy. To investigate this, we disrupted secretory autophagy by inhibiting the GTPase RhoA and its downstream effector Rho kinase (ROCK), which were previously shown to promote autophagosome formation. Treatment of MKs with inhibitors of autophagy or RhoA/ROCK markedly reduced TGFβ1 secretion in vitro and led to intracellular accumulation of TGFβ1. Taken together, these data suggest that TGFβ1 is secreted by MKs via secretory autophagy. To examine whether secretory autophagy in MKs contributes to disease pathogenesis in vivo, we employed an MPL W515L-driven transplant model. Basal autophagy measured by LC3B conversion and RhoA protein levels were increased in platelets derived from mice with MPL W515L-driven MF. Conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system ( Mx1-Cre+; Atg5fl/fl mice) prevented leukocytosis, MK clustering and aberrant TGFβ1 secretion in the MPL W515L-driven transplant model, indicating that autophagy promotes the initiation of MF. Furthermore, MPL W515L-transplanted mice treated with a low-dose ROCK inhibitor (ROCKi) - several of which are FDA-approved - reduced MK clustering and fibrosis in spleens and femurs of treated mice (Figure 1a, b). To further verify our data, we utilized a second preclinical mouse model of MF - a knockout mouse lacking the inhibitory MK receptor G6b-B ( Mpig6b -/-), which exhibits thrombocytopenia, MK clustering and MF. In line with our previous findings, conditional deletion of Rhoa in MK lineage cells in Pf4-Cre+; Rhoafl/fl; Mpig6b -/- mice normalized TGFβ1 levels within the bone marrow and protected against the development of fibrosis in bone marrow and spleen. In ongoing work, ROCK inhibitors are further being tested in higher doses alone or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data suggest that TGFβ1 secretion is uncoupled from conventional granule secretion in MKs and is instead dependent on RhoA/autophagy signaling pathways. Targeting these pathways in aberrant MKs represents a new potential therapeutic target in the treatment of MF.

Identifying Targeted Therapies for CBFA2T3- GLIS2 Acute Myeloid Leukemia

CBFA2T3- GLIS2 fusion positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. Children with this subtype of AML frequently experience upfront chemoresistance with a high cumulative incidence of relapse (42-83%) and a dismal overall survival (~20%) even with intensive therapy. New treatment approaches are urgently needed. By interrogating the Broad Institute's Cancer Dependency Map (DepMap), a data set composed of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, we discovered that the CBFA2T3- GLIS2 AML model M-07e was highly dependent on the kinase JAK2. Indeed, M-07e was one of the most JAK2 dependent models in the data set. Using the same genome-scale library, we next performed CRISPR-Cas9 screens in WSU-AML and CMS, two additional AML cell lines harboring this fusion, and revealed JAK2 as a strong dependency shared by the 3 cell lines. Using a doxycycline-inducible knockout (KO) system, we validated JAK2 KO by western blot in three AML cell lines and cells from a patient-derived xenograft (PDX) harboring the fusion. Using two different sgRNAs targeting JAK2 (sgJAK2) and a non-targeting (sgNT) control, we validated JAK2 dependency in all 4 AML models in competitive growth assays. We also observed impaired proliferation and induced apoptosis upon JAK2 KO. To evaluate a role for JAK2 dependency in vivo, we intravenously injected NSGS mice with WSU-AML either transduced with doxycycline-inducible sgJAK2 or sgNT. After confirmation of engraftment by bone marrow evaluation, doxycycline chow was initiated. After 2 weeks of doxycycline treatment, we observed a significantly decreased tumor burden in mice with JAK2 KO AML compared to sgNT as measured by a decrement in human CD45% cells: peripheral blood (2.8% versus 12.9%), bone marrow (37.4% versus 56.3%,) and spleen (19% versus 79.5%). We next assessed chemical inhibitors of JAK2. Both the type I JAK2 inhibitor ruxolitinib and the type II JAK2 inhibitor CHZ868 exhibited strong activity in vitro in CBFA2T3- GLIS2 cell lines and PDX cells, many resistant to cytotoxic chemotherapy. Because ruxolitinib is FDA-approved for other indications, we evaluated this drug in a cell line and PDX model of CBFA2T3- GLIS2 AML in vivo. As previously reported in myeloproliferative neoplasms, JAK2 target inhibition with ruxolitinib treatment was incomplete, and accordingly, in vivo response was modest with leukemia ultimately progressing. Evaluation of CHZ868 in vivo is ongoing. Because single agent targeted therapy is typically insufficient for durable response in the treatment of most cancers, we utilized CRISPR-Cas9 anchor screening to identify candidate resistance mechanisms and potential synergistic combinations with JAK2 inhibitors in CMS and WSU-AML cells. We compared the changes in abundance of sgRNAs in the cells either treated with ruxolitinib (IC 50) or vehicle for 14 days. The correlation of hits between the two cell lines was strong, and we found that sgRNAs targeting multiple negative regulators of the MAPK pathway were significantly enriched in the ruxolitinib-treated arm. In parallel, we generated resistant CMS sublines by chronic exposure to ruxolitinib. Ruxolitinib-resistant CMS cells were found to have a pathogenic NRAS missense mutation and an associated increase in levels of phosphorylated MEK. Accordingly, they were responsive to the MEK inhibitor trametinib. Both approaches converge on activation of the MAPK pathway as a resistance mechanism to JAK2 inhibitors in CBFA2T3- GLIS2 AML. We next hypothesized that targeting the MAPK pathway could overcome resistance to JAK2 inhibitors in this disease context. Indeed, treatment with ruxolitinib and the MEK inhibitor trametinib exhibited a synergistic effect when used in combination in cell lines and PDX cells harboring the fusion in vitro with in vivo testing as a next step. In summary, we validated JAK2 as a strong dependency in CBFA2T3- GLIS2 fusion positive pediatric AML. We demonstrated that JAK2 KO or small molecule inhibitors impaired cell viability and induced apoptosis in vitro, and JAK2 KO was highly efficacious in decreasing leukemia burden in vivo. Activation of the MAPK pathway renders resistance to JAK2 inhibitors and the combination of a JAK2 with MEK inhibitor is highly synergistic.

Defective Clearance of Neutrophils Causes Pathogenic Microenvironmental Interactions and Offers a Candidate Immunotherapy in the Myeloproliferative Neoplasms

Neutrophils are the most abundant leukocytes and essential components of the immune microenvironment. Myeloproliferative neoplasms (MPNs) are characterized by excessive myelopoiesis and serious complications, such as thrombosis, myelofibrosis and leukemic transformation. Neutrophils and hematopoietic stem cells (HSCs) carrying the most common MPN driver mutation (JAK2 V617F) are protected from apoptosis. Moreover, mutant neutrophils aggregate with platelets and interact with activated endothelium, increasing thrombosis risk in MPN (Blood 107:3676; Thromb Res 129:263; Sci Transl Med 10:eaan8292; Leukemia 33:2544). However, possible pathogenic interactions between heterogeneous bone marrow (BM) niches in MPN (Nat Cancer doi:10.1038/s43018-023-00607-x) and neutrophils are understudied. Circulating HSCs and leukocytes home daily to the BM (Nature 452:442), where apoptotic/senescent neutrophils are cleared by macrophages (Immunology 125:281; Cell 153:1025). We found that senescent neutrophils carrying JAK2 V617F or CALR del52 mutation evade normal clearance and accumulate in the BM of MPN mice. The homeostatic recognition and efferocytosis of neutrophils by macrophages rely on the balance of “eat-me” and “don't-eat-me” signals. Supervised analyses of two independent human JAK2 V617F+ MPN granulocyte RNAseq datasets (Blood 123:e123; Blood 134:199) showed specific upregulation of CD24 - reportedly a don't-eat-me signal in solid tumors (Nature 572:392). CD24 expression was high in granulocytes from MPN transformed into AML and in the JAK inhibitors ruxolitinib and fedratinib-resistant HEL cells (Nat Cancer 4:108). Unlike CD274 or CD47, which are extensively studied in myeloid malignancies, our data showed that CD24 expression was high in JAK2 V617F+ human or mouse neutrophils. Functional blockade in co-culture with macrophages or after in vivo adoptive transfer showed that CD24 serves as a don't-eat-me signal in neutrophils. Given that oncogenic driver mutations lead to the constitutive activation of cytokine-regulated JAK-STAT signaling, we investigated the effect of cytokines activating this pathway. We found that high GM-CSF in MPN BM induces CD24 expression and prevents neutrophil clearance via JAK-STAT5 signaling. Consequently, ruxolitinib normalized GM-CSF-induced CD24 expression and decreased human and mouse neutrophils in MPN. Our study showed that accumulation of mutant senescent neutrophils in the BM triggered abnormal interactions within the BM microenvironment. Mutated neutrophils were found closer to Megakaryocytes (Mk) and ~30% of Mks showed neutrophils transiently invading their demarcation membrane system through a process termed “emperipolesis”, which increases thrombopoiesis and intercellular exchange between neutrophils and Mks (Elife 8:e44031) and frequently occurs in diseases associated with myelofibrosis, such as MPN (Blood 104:3573) and grey platelet syndrome (Blood 124:3624). To examine whether mutated neutrophils affect platelet production, we transferred wild-type or JAK2 V617F+ senescent neutrophils and found that JAK2 V617F+ mutated (but not WT) senescent neutrophils increased Mk emperipolesis and circulating platelets. Supervised RNA-seq analysis of CD24 hi JAK2 V617F+ human neutrophils highlighted their pro-inflammatory signature. Furthermore, CD24 expression positively correlated with neutrophil degranulation signatures and proteolytic enzymes that activate latent pro-fibrotic TGF-β in Mk, subsequently triggering myelofibrosis. Taken together, these findings suggest that accumulation of senescent neutrophils might lead to their pathogenic interactions with Mks or platelets, which may increase thrombocytosis and myelofibrosis risk in MPN. To test this hypothesis and the therapeutic potential of CD24 inhibition, MPN mice were chronically treated with CD24 blocking antibody. This treatment improved thrombocytosis, decreased platelet-neutrophil complexes, restored normal clearance of mutated senescent neutrophils and reduced emperipolesis levels which, in turn, decreased active TGF-β, ultimately improving myelofibrosis. This study reveals defective neutrophil clearance as a cause of pathogenic microenvironmental interactions that may increase thrombosis and myelofibrosis risk, and postulate CD24 as a candidate target for innate immune checkpoint in myeloid malignancies.

BCL-Xl Represents a Novel Therapeutic Target in Type 2 Mutant Calr-Driven Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal disorders that arise in the hematopoietic stem cell (HSC) compartment and that result in the excess production of mature blood cells of the myeloid lineage. All MPNs are unified by their reliance on the constitutive activation of the JAK/STAT pathway. Nevertheless, JAK inhibitors have proven insufficient to significantly improve disease outcome, highlighting the need to further understand the molecular mechanisms driving these diseases and to discover novel therapeutic targets. Calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, has been found to be mutated in ~40% of essential thrombocytosis (ET) and myelofibrosis (MF) cases. The most common mutations in CALR are type 1 (52 base pair deletion; CALRdel52) and type 2 (5 base pair insertion; CALRins5) mutations, which both confer distinct clinical and prognostic characteristics. We have previously demonstrated that mutant CALR-driven MPNs rely not only on aberrant JAK/STAT signaling but also on the upregulation of BCL-2 family proteins and activation of the unfolded protein response (UPR) pathway (ASH abstract #147859, 2021). Specifically, we have shown that CALRins5 mutations lead to activation of and dependency on the activating transcription factor 6 (ATF6) branch of the UPR. In parallel, we found that CALRins5 cells exhibit differential up-regulation of the anti-apoptotic protein BCL-xL, but not BCL-2 (ASH abstract #3588, 2021). Based on these findings, we hypothesized that the active form of ATF6, which functions as a transcription factor, may directly transcriptionally activate BCL-xL leading to its up-regulation specifically in CALRins5 but not CALRdel52 MPN cells, and that the ATF6/BCL-xL axis may represent an important pro-survival mechanism that can be targeted for therapeutic gain in CALRins5 MPN patients. To test this hypothesis, we first validated that BCL-xL is significantly up-regulated in CALRins5 versus CALRdel52 and CALR wild type (CALRwt) human cell lines and peripheral blood mononuclear cells (PBMCs) from MPN patients. Because BCL-xL can also be regulated downstream of JAK/STAT signaling, we next tested whether the increased expression of BCL-xL in CALRins5 cells is due to ATF6 activation, JAK/STAT activation, or both. In cells treated with JAK inhibitor ruxolitinib, we found that while BCL-xL expression levels decreased nearly 90% in ruxolitinib treated CALRdel52 cells, this decrease was much more modest in CALRins5 cells, suggesting JAK/STAT activation is only partially responsible for BCL-xL up-regulation in CALRins5 MPN cells. Conversely, in ATF6 knockout cells, we found that deletion of ATF6 did not affect BCL-xL levels in CALRwt or CALRdel52 cells, but did significantly abrogate BCL-xL up-regulation in CALRins5 cells. This suggests that in CALRins5 cells, while JAK/STAT signaling contributes in part to BCL-xL up-regulation, ATF6 is the key mediator of BCL-xL expression. CUT&amp;RUN studies further revealed that ATF6 directly regulates BCL-xL transcription. Lastly, we sought to test whether inhibition of BCL-xL alone is sufficient to induce apoptosis in CALRins5 cells. BH3 profiling studies revealed that CALRins5 cells are more primed for apoptosis via BCL-xL peptide antagonism compared to CALRdel52 cells. We next evaluated the activity of A-1331852, a BCL-xL selective inhibitor, alone or in combination with ruxolitinib, in human cell lines and PMBCs from MPN patients. We observed that CALRins5 cells displayed decreased viability in response to A-1331852 alone and in combination treatments, suggesting an increased sensitivity to BCL-xL inhibition. In conclusion, we demonstrate that CALRins5-driven MPN cells display an enhanced sensitivity to BCL-xL inhibition, which may represent an effective therapeutic approach for CALRins5+ MPN patients.