Combination Of Ruxolitinib Research Articles

Combination ruxolitinib and belumosudil is tolerable and induces responses despite treatment failure as monotherapies

FDA approved agents for the treatment of chronic GVHD including ruxolitinib and belumosudil are effective steroid-sparing agents, with overall response rates (ORR) of 76% and 65% respectively. Ruxolitinib and belumosudil are well tolerated with different primary targets. Little data is available on the use of combination ruxolitinib and belumosudil. This is a single center, retrospective analysis of 20 treatment-refractory patients with chronic GVHD treated with combination ruxolitinib and belumosudil. The ORR including complete response (CR) and partial response (PR) at any time was 55% (11/20). Among responding patients, other immunosuppressive agents were tapered or discontinued in all patients. None of the patients developed EBV or CMV reactivation requiring treatment. 4 patients (20%) developed pneumonia and 2 patients (10%) developed viral URI. Cytopenias were not exacerbated. No patients had graft failure or relapsed disease. The combination is tolerable, delays the need for alternative therapies, and facilitates tapering of corticosteroids.

Combination of JAKi and HDACi Exerts Antiangiogenic Potential in Cutaneous T-Cell Lymphoma.

Angiogenesis plays a pivotal role in the growth and metastasis of tumors, including the development and progression of cutaneous lymphomas. The chick embryo CAM model has been utilized in various studies to assess the growth rate, angiogenic potential, and metastatic capability of different tumor types and malignant cell lines. However, the precise mechanisms of angiogenesis in CTCL and the influence of Ruxolitinib or Resminostat on angiogenesis in hematological malignancies and solid tumors are not well understood. Recent in vitro and in vivo data have demonstrated the synergistic inhibition of tumorigenesis and metastasis in experimental models of CTCL when using the combination of Resminostat (HDACi) with Ruxolitinib (JAKi). The present work aims to elucidate the effects of this combination on the tumor microenvironment's vascular components. We investigated the effects of Ruxolitinib (JAKi) in combination with Resminostat (HDACi) treatment in transendothelial migration of CTCL cells (106 MyLa and SeAx) by using a transwell-based transendothelial migration assay and tumor angiogenesis in vivo. We used the CTCL chick embryo CAM model with xenografted tumors derived from implanted MyLa and SeAx cells and administered topically 15 μM ruxolitinib and 5 μM Resminostat every two days during a 5-day period. JAKi and HDACi inhibited CTCL cell transendothelial migration by 75% and 82% (p < 0.05) in both CTCL engrafted cells (MyLa and SeAx, respectively) compared to the untreated group. Moreover, the combination of ruxolitinib with resminostat blocked angiogenesis by significantly reducing the number of blood vessel formation by 49% and 34% in both MyLa and SeAx, respectively (p < 0.05), indicating that the proposed combination exerted significant anti-angiogenic effects in the CAM CTCL model. Overall, these data provide valuable insights into potential therapeutic strategies targeting angiogenesis in CTCL, paving the way for more effective treatment approaches in the future.

A Phase I Trial Evaluating the Addition of Lenalidomide to Patients with Relapsed/Refractory Multiple Myeloma Progressing on Ruxolitinib and Methylprednisolone.

Ruxolitinib (RUX), an orally administered selective Janus kinase 1/2 inhibitor, has received approval for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. We have previously demonstrated the anti-multiple myeloma effects of RUX alone and in combination with the immunomodulatory agent lenalidomide (LEN) and glucocorticosteroids both pre-clinically and clinically. This study aims to evaluate whether LEN can achieve clinical activity among patients with multiple myeloma progressing on the combination of RUX and methylprednisolone (MP). In this part of a phase I, multicenter, open-label study, we evaluated the safety and efficacy of RUX and MP for patients with multiple myeloma with progressive disease who had previously received a proteasome inhibitor, LEN, glucocorticosteroids, and at least three prior regimens; we also determined the safety and efficacy of adding LEN at the time of disease progression from the initial doublet treatment. Initially, all subjects received oral RUX 15 mg twice daily and oral MP 40 mg every other day. Those patients who developed progressive disease according to the International Myeloma Working Group criteria then received LEN 10 mg once daily on days 1-21 within a 28-day cycle in addition to RUX and MP, which were administered at the same doses these patients were receiving at the time progressive disease developed. Twenty-nine subjects (median age 64 years; 18 [62%] male) were enrolled in this part of the study and initially received the two-drug combination of RUX and MP. The median number of prior therapies was six (range 3-12). The overall response rate from this two-drug combination was 31% and the clinical benefit rate was 34%. The best responses were 1 very good partial response, 8 partial responses, 1 minor response, 12 stable disease, and 7 progressive disease. The median progression-free survival was 3.5 months (range 0.5-36.2months). The median time to response was 3.0 months. The median duration of response was 12.5 months (range 2.8-36.2 months). Twenty (69%) patients whoshowed progressive disease had LEN added to RUX and MP; all patients had prior exposure to LEN and all but one patient was refractory to their last LEN-containing regimen. After the addition of LEN, the overall response rate was 30% and the clinical benefit rate was 40%. The best responses of patients following the addition of LEN were 2 very good partial responses, 4 partial responses, 2 minor responses, 8 stable disease, and 4 progressive disease. The median time to response was 2.6 months (range 0.7-15.0 months). The median duration of response was not reached. The median progression-free survival following the addition of LEN was 3.5 months (range 0.3-25.9 months). For patients with multiple myeloma, treatment with RUX and MP is effective and well tolerated, and LEN can be used to extend the benefit of this RUX-based treatment. This study is registered with ClinicalTrials.gov, NCT03110822, and is ongoing.

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.

Abstract 7225: Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms

Abstract Myeloproliferative neoplasms (MPNs) are characterized by aberrant activation of the JAK-STAT pathway. Ruxolitinib (RUX) is a JAK1/2 inhibitor used to treat myelofibrosis (MF) and hydroxyurea-resistant/intolerant polycythemia vera. While RUX has demonstrated important clinical benefits, a large proportion of patients have persistent disease manifestations despite therapy. CDK8 regulates phosphorylation of STAT proteins in a RUX-independent manner; thus, CDK8 inhibition may alter downstream STAT target gene expression. RVU120 is a highly selective and potent CDK8/19 inhibitor that inhibits STAT1/5 phosphorylation. We hypothesized that the combination of RUX and RVU120 would act in a cooperative manner to attenuate JAK-STAT signaling and reduce MPN phenotypes in vivo. Using JAK2V617F mutant leukemic cell lines, we identified dose-responsive changes in viability with RUX and RVU120 in all cell lines. Pharmacodynamic analysis validated that STAT5 protein phosphorylation was decreased by RUX/RVU120, although at different phosphorylation sites by RUX (pTYR694) and RVU120 (pSER726/731); exposure to RUX+RVU120 inhibited phosphorylation at both sites. Notably, analysis of combinatorial effects of RUX+RVU120 on viability demonstrated drug synergies (combinatorial index &amp;lt;1) in RUX-naïve and resistant SET-2 cells. Nascent RNA expression data exploring JAK-STAT and mediator-related pathways of pathogenesis will be presented at the meeting. For in vivo studies, we used two previously-described murine models of MF and PV. Briefly, retrovirally-transfected (MSCV-MPLW515L-IRES-GFP) or Jak2V617F-conditional knock-in bone marrow (BM) was intravenously injected into lethally-irradiated congenic recipients. Mice were randomized and treated for several weeks with vehicle (VEH), single-agent RUX/RVU120, or RUX+RVU120 arms. Spleen weights between VEH and RUX/RVU120 treated mice at time of terminal sacrifice were significantly reduced across both models (p⇐0.009). In MPLW515L mice, WBCs and PB GFP% were significantly reduced (p&amp;lt;0.002) and histopathology demonstrated near-elimination of BM reticulin fibrosis and improved trilineage hematopoiesis between RUX+RVU120 and RUX arms. Data on overall survival, cytokine profiles, and the impact of RVU120 on dynamic gene expression will be presented at the meeting. The combination of RVU120 and RUX demonstrated biochemical synergy and differential inhibition of STAT5 phosphorylation in vitro. Further, in vivo treatment with RVU120/RUX+RVU120 resulted in significant reductions of disease manifestation (splenomegaly, WBC, fibrosis scoring, hematopoiesis) when compared to VEH/RUX. These data nominate JAK1/2 and CDK8/19 inhibition as a potential novel therapeutic strategy in MPNs; further work on nascent RNA expression and cytokine profiles will potentially elucidate additional mechanisms of action. Citation Format: Zachary Zaroogian, Elżbieta Adamczyk, Adrianna Moszyńska, Marta Obacz, Urszula Pakulska, Benjamin Durham, Milena Mazan, Shoron Mowla, Katarzyna Wnęk, Amritha Varshini Hanasoge Somasundara, Beata Barczuk, Aniela Gołas, Ross Levine, Tomasz Rzymski, Raajit Rampal. Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7225.

Combination Ruxolitinib and Belumosudil Is Tolerable and Induces Responses Despite Treatment Failure As Monotherapies

Combination Ruxolitinib and Belumosudil Is Tolerable and Induces Responses Despite Treatment Failure As Monotherapies

Therapeutic Targeting of JAK/STAT Signaling and Histone Acetyltransferase Activity in Post-Myeloproliferative Neoplasm Secondary AML

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling is present in 50-90% of MPNs, underscoring its role in driving MPN pathogenesis and transformation to acute sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. Despite enhancing patients' quality of life, ruxolitinib did not exert major improvements on disease outcome (Eghtedar et al, 2012). Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CREBBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML using high throughput genetic screens, in vitro functional assays, transcriptomic and epigenetic profiling and mouse xenograft in vivo experiments. To identify druggable determinants of JAK2 inhibitor response in post-MPN sAML, we performed genome-wide loss-of-function CRISPR screens in sAML HEL cells treated with four different JAK2 inhibitors, including ruxolitinib. We discovered that depletion of the histone acetyl transferase CREBBP sensitizes HEL cells to JAK2 inhibition (Figure 1A). Pharmacological inhibition of JAK/STAT signaling and HAT activity using a combination of ruxolitinib and CREBBP inhibitors significantly reduced the proliferation of sAML cells HEL and SET2 (Figure 1B). Among the CREBBP inhibitors, CBP30 synergizes best with ruxolitinib having Chou-Talalay combination indices of 0.152 and 0.704 in HEL and SET2, respectively. Moreover, combination treatment of ruxolitinib and CBP30 markedly increased apoptosis and induced cell cycle arrest at G1 in HEL. To mechanistically characterize the synergism between ruxolitinib and CBP30, we performed ChIP-seq to profile STAT5 binding and H3K27ac levels in HEL cells treated with DMSO, ruxolitinib, CBP30 and their combination. STAT5 binding profiles did not reveal major differences in ruxolitinib versus ruxolitinib + CBP30 treated cells, suggesting that the drug combination does not significantly alter STAT5 binding. Conversely, we noticed striking global changes in H3K27ac levels in the combination compared with DMSO or single agents, indicating that the synergistic effect between ruxolitinib and CBP30 is mediated by substantial changes in transcriptional regulation. Notably, analysis of H3K27ac downregulated and upregulated sites identified enrichments in motifs recognized by FOS/SMAD4 and GATA transcription factors, respectively. These TFs are involved in the regulation of myeloid differentiation implicating a synergistic regulation of relevant myeloid differentiation pathways by the combination of JAK2 and CREBBP inhibitors in sAML. Furthermore, ruxolitinib + CBP30 treatment in HEL elicits a unique transcriptome profile with expression deregulation of genes associated with cell cycle, DNA repair, HAT activity and TGF-beta signaling as determined by DAVID functional annotation analysis. Lastly, to evaluate the synergistic effects of ruxolitinib and CBP30 in vivo, we treated luciferase-expressing HEL sAML mouse xenografts with vehicle, ruxolitinib (80 mg/kg), CBP30 (30 mg/kg) and ruxolitinib (80 mg/kg) + CBP30 (30 mg/kg). Combination of ruxolitinib and CBP30 displayed significant decrease in leukemia burden after 4 weeks of treatment as monitored by bioluminescence imaging and quantification. Consistently, mice treated with both drugs also showed remarkable decrease in their white blood cell count and spleen size compared with single-agent-treated and vehicle-treated mice. Collectively, our results substantiate the combinatorial therapeutic targeting of JAK/STAT signaling and HAT activity as a potential treatment approach for post-MPN sAML.

Evaluation of Safety and Efficacy of Ruxolitinib in Patients with Pediatric Lymphoproliferative Disorders

Introduction. Pediatric lymphoproliferative disorders (PLPD) encompass a wide spectrum of immune dysregulation conditions with heterogeneous clinical presentation and lab abnormalities. The underlying mechanisms of PLPD are diverse, including dysregulated differentiation of immune cell populations, aberrant recruitment of lymphocyte populations, and/or retention and pathologic survival of lymphocytes. Many PLPD are associated with inborn or acquired genetic factors impacting immune function. Standardized measures to characterize clinical and biological factors in PLPD patients and responses to therapy are lacking. One common feature in PLPD patients is activating IFNg signaling. Ruxolitinib is a drug that blocks hyperactivated IFNg signaling through the JAK-STAT pathway. The goal of this study is to describe the clinical responses of PLPD patients to ruxolitinib. Methods. Medical charts were reviewed to identify patients with PLPD treated with ruxolitnib at Texas Children's Hospital over a 7-year period. Clinical manifestations (recurrent fever, fatigue, lymphadenopathy, recurrent infections, pulmonary disease, endocrinopathy, enteropathy, hepatosplenomegaly, arthritis, CNS symptoms, atopy, dysphagia, underweight), lab values of complete blood count and pathologic inflammation markers (CXCL9, ferritin, sIL2R, IL-18, CD163, neopterin) were categorized with a severity system indicating: 0 no clinical signs and normal lab values; 1 mild to moderate symptoms and abnormal lab values; 2 severe symptoms, highly abnormal lab values (table 1). Total score was computed before and during the addition of ruxolitinib. Complete response (CR) represented a reduction of the total score to 0. Partial response (PR) signified reduction of criteria score ≥ 30% from the baseline with no progression or manifestation of new symptoms. Progression of the disease or improvement &amp;lt; 30% was marked as no response (NR). Results. Chart review identified 19 children and young adults with PLPD who received treatment with ruxolitinib (median age 8.5 years; range 1.2 to 21.6 years). Underlying disease states driving PLPD included HLH, inborn errors of immunity, juvenile rheumatoid arthritis, macrophage activation syndrome, malignant atrophic papulosis of Degos, Aicardi-Goutierres Syndrome, and T-cell large granular lymphocytic leukemia.Genetic variants known or predicted to be damaging were identified in 15 cases (79%), including STAT1, STAT3, TREX1, OFD1, NLRP12, NLRP4, UNC13, PRF1, AP3D1 and mosaic genome-wide paternal uniparental disomy. While 79% of patients received ruxolitinib in combination with other immune suppressants, 21% received only ruxolitinib out of which half received it as front-line therapy. Based on the PLPD Scoring System, overall response rate to ruxolitinib was 73.7%, with 5.3% achieving CR, and 68.4% PR. Overall survival on this study was 84.2% with median 11 months follow-up (range: 0.5 month to 7.1 years). Three patients died due to uncontrolled disease progression. Six patients in whom hematopoietic cell transplant was deemed essential for cure survived to conditioning. Toxicities potentially related to ruxolitinib included grades 1 to 4 cytopenias and grade 1 elevated liver enzymes. Conclusion. PLPD represents a population with overall poor outcomes. We developed standardized staging and response metrics to characterize extent of immune dysregulation and response to therapy. Ruxolitinib represents a potential “precision” therapeutic intervention for patients with pathologic IFNg pathway activation. Ruxolitinib was well-tolerated in this high risk population, and was associated with positive responses in the majority of patients. This study supports expansion of prospective studies to validate the PLPD Scoring System and to optimize therapeutic strategies to manage immune dysregulation, including ruxolitinib.

The Novel Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) Platform Identifies Synergistic Activity of Lanraplenib and Ruxolitinib in Hematological Malignancies

Background: Investigation of drug combinations across different contexts can provide useful insights on the anti-cancer mechanism and can ultimately lead to new treatments. However, conventional drug combination screening methods are limited by throughput. Efforts to systematically identify the most effective active combinations and the optimal molecular contexts in a high throughput screening (HTS) format could greatly accelerate the development of combination treatments. Spleen Tyrosine Kinase, SYK, is a non-receptor tyrosine kinase known to regulate intracellular signaling, including FLT3, AKT/mTOR and STAT5 pathways, via its immunoreceptor tyrosine-based activation motif (ITAM). Deregulated SYK signaling has been reported to play a central role in pathogenesis of allergic and autoimmune diseases as well as hematological malignancies. Lanraplenib (LANRA) is a next-generation SYK inhibitor currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) (NCT05028751). Given its critical role in intracellular signaling and interaction with receptor tyrosine kinases(RTKs), we hypothesized that LANRA could synergize with ruxolitinib, a JAK inhibitor. To address this hypothesis, we performed a high throughput drug combination screen using the Broad Institute's Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) platform, which enables rapid screening of thousands of compounds in a 930-cell line panel across 45 different lineages. Methods: In this study, we performed a combination screen using the PRISM platform with ruxolitinib as the test compound, with a 10 μM top dose (7 dose concentrations with 3-fold dilutions), in combination with LANRA at two anchor doses, 2 μM and 10 μM. PRISM is a pooled, multiplexed cell viability assay that provides 7-point dose response curves, IC 50, AUC values, and relative abundance of unique cell line barcodes. To understand the drug synergy landscape across different lineages, we developed a bioinformatics pipeline which uses PRISM viability data to calculate synergy scores across all the cell lines and drug combinations. Secondary validation studies of the combinations used CTG viability measurements. Phospho-SYK expression was evaluated in archival formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with myeloid proliferative neoplasm (MPN) by immunohistochemistry (IHC). RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. Results: In the PRISM cell line panel, ruxolitinib in combination with LANRA demonstrated synergistic activity in hematological malignancy cell lines. Among AML cell lines in the panel, OCIAML5, OCIM1, HL60, HEL, EOL1, MONOMAC6, NB4, U937, PL21, and TF1 showed the highest synergy scores. The most sensitive cell lines to the combination showed up-regulation of JAK-STAT and inflammatory signaling pathways in a gene set enrichment analysis (GSEA) prior to treatment. Consistent with this, phosphorylated SYK was associated with inflammatory megakaryocytes and fibrosis in primary samples from patients with MPN. LANRA down-regulated JAK-STAT signaling in a reporter cell line and repressed gene expression associated with dysregulated inflammatory pathways in AML cells. Additionally, the combination of LANRA and ruxolitinib showed synergistic antiproliferative activity across a broad range of concentrations in FLT3-ITD primary AML cells and a panel of hematological malignancy cell lines, confirming the PRISM results. Conclusion: These studies demonstrate the utility of PRISM as a platform to rapidly identify rational combination agents. Importantly, LANRA is effective in combination with ruxolitinib in AML and other hematological malignancy preclinical models. This finding is consistent with the observation that SYK can regulate STAT signaling and cooperate with other RTKs like FLT3. Given the central role of SYK in regulation of oncogenic and inflammatory signaling, SYK inhibition with LANRA in combination with ruxolitinib may be a promising strategy for patients with myeloid malignancies.

A Phase I Study of Ruxolitinib in Combination with Abemaciclib for Patients with Primary or Post-Polycythemia Vera/Essential Thrombocythemia Myelofibrosis

Background and Significance: Patients (pts) with primary or secondary myelofibrosis (MF) experience variable degrees of constitutional symptoms, cytopenias and risk of progression to acute myeloid leukemia leading to a reduced life-expectancy compared to age-matched controls. Although several oral Janus Kinase (JAK) inhibitors such as ruxolitinib (RUX) have been approved by the US FDA based on improvements in symptom burden and spleen volume compared to physician's choice of best available therapy, the duration of response to these agents is generally time-limited and treatment with JAK inhibitors is not considered to have disease-modifying potential. Furthermore, the median overall survival after RUX discontinuation is only 11-14 months highlighting the need for novel mechanism-based therapies for pts with disease progression on RUX. JAK2 V617F mutations promote transition from the G1 to S-phase of the cell cycle via increased expression of CDC25A, which is also upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been implicated in MF pathogenesis and contribute to myeloproliferation. Finally, we and others have shown synergistic effects of the combination of CDK4/6 inhibitors and RUX in murine models of MF (Rampal et al., CCR 2021) including improvement in bone marrow fibrosis suggesting the potential for disease-modifying activity. Several CDK4/6 inhibitors have been approved by the US FDA for the treatment of hormone receptor-positive metastatic breast cancer establishing a robust record of safety data. These preclinical data and known safety profile of CDK4/6 and RUX as single agents, provide rationale for combining CDK4/6 and JAK inhibition in pts with MF. Methods: This multicenter, phase I dose-escalation trial (NCT05714072) evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in primary or secondary MF pts with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. Inadequate response to RUX is defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms, as defined by the presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS. Pts must have been on a stable dose of 10 mg or 15 mg BID of RUX for at least 4 weeks prior to enrollment with adequate platelet (≥75 x 10 9/L) and neutrophil counts (≥1.5 x 10 9/L). Key exclusion criteria include prior treatment with a CDK4/6 inhibitor, accelerated- or blast-phase MPN, platelet count &amp;lt;50 x 10 9/L in the 4 weeks before screening or platelet transfusion(s) within 8 weeks before screening, and absolute neutrophil count &amp;lt;0.5 x 10 9/L in the 4 weeks before screening. Among the currently FDA-approved CDK4/6 inhibitors, abemaciclib was chosen for combination therapy due to less myelosuppressive effects compared with other CDK4/6 inhibitors. This trial uses a conventional “3+3” dose-escalation design ( Figure), pts will be treated with increasing doses of abemaciclib added to a stable dose of RUX (10 mg or 15 mg BID). The pre-study dose of RUX will be maintained to determine the maximum tolerated dose of the combination of RUX and abemaciclib. The primary endpoint is to determine the maximum tolerated dose of RUX + abemaciclib. Secondary endpoints include measures of efficacy such as overall response rate, duration of response and survival endpoints. Exploratory endpoints include changes in allele fraction of driver mutations, gene expression profiling, and clonal architecture to identify biomarkers of response, mechanisms of resistance, and to assess for any disease-modifying effects of the combination therapy. At this time, 2 pts have been enrolled at dose level 0 without any dose-limiting toxicities observed. In line with the known adverse event profile of abemaciclib, grade 1 diarrhea occurred in one patient. One patient developed grade 3 neutropenia which promptly resolved with holding abemaciclib. No unexpected safety signal was noted and enrollment continues. This trial is open to enrollment at Memorial Sloan Kettering Cancer Center and will commence at MD Anderson Cancer Center later this year.

ALK2 and JAK2 Inhibition for Improved Treatment of Anemia in Myelofibrosis Patients: Preclinical Profile of an ALK2 Inhibitor Zilurgisertib in Combination with Ruxolitinib

Introduction: Anemia is a common occurrence in patients with myelofibrosis (MF) and is associated with the need for red blood cell transfusion and poor clinical prognosis. JAK inhibitors such as ruxolitinib are used extensively to treat symptoms of MF and improve quality of life and overall survival. However, JAK inhibitors may also contribute to myelosuppression. Therapeutic interventions that allow for optimization of JAK inhibition with no concern of anemia would benefit patients with MF and potentially lower the rate of treatment discontinuation or suboptimal dosing. Recent studies have demonstrated that inhibition of ACVR1/ALK2, a bone morphogenetic protein receptor that is upstream of the transcriptional regulation of hepcidin, could reduce serum hepcidin levels in patients with MF and improve anemia (Verstovsek S et al. Lancet 2023; Oh S et al. Clin Lymphoma Myeloma Leuk 2022). Reducing levels of hepcidin, a key regulator of plasma iron levels, and restoring erythropoiesis would broadly benefit patients with MF. Based on these recent data, we developed zilurgisertib, a potent and selective ALK2 inhibitor that could be administered in combination with ruxolitinib at doses titrated to the needs of patients with MF. Methods and Results: In biochemical and cellular assays, zilurgisertib inhibited ALK2 kinase activity and SMAD1/5 phosphorylation with IC 50 values of 15 nM and 63 nM, respectively. In Huh-7 cells stimulated with BMP-6, zilurgisertib inhibited hepcidin production with an IC 50 of 20 nM, demonstrating the compound is a potent ALK2 inhibitor capable of regulating iron homeostasis via hepcidin. To assess possible off-target effects of zilurgisertib, kinome profiling at 10 μM ATP was performed at Reaction Biology (Malvern, PA) to determine the overall specificity across 356 kinases (see Figure). At 200 nM, zilurgisertib only inhibited ALK2, ALK1 (to 50%), and ALK6 (to 48%). In addition, zilurgisertib at 20 μM did not affect viability of HEK293 cells, a human cell line commonly used to assess general cell health and compound toxicity. Similarly, zilurgisertib did not affect viability of human fibroblasts or endothelial cells at concentrations up to 5 μM. To test whether the combination of ruxolitinib and zilurgisertib could suppress hepcidin and restore erythropoiesis in an in vivo mouse model of cancer-induced anemia, B16F10 cells were injected intraperitoneally, mimicking a metastatic tumor that leads to anemia 1 week after injection. In a dose-dependent manner, zilurgisertib improved hemoglobin by 2 to 3 g/dL and red blood cell counts, while reducing both liver pSMAD and circulating hepcidin levels by 50% or more compared with vehicle control. The combination of zilurgisertib with ruxolitinib had no effect on this activity, suggesting that inhibition of JAK2 does not inhibit erythropoiesis restored following ALK2 inhibition. Conclusion: Taken together, the potent and selective on-target activity of zilurgisertib suggests that ALK2 inhibition could reduce hepcidin and improve anemia, and that the combination of zilurgisertib with ruxolitinib is a rational and attractive approach to mitigate anemia in patients with MF. The combination of ruxolitinib and zilurgisertib is currently being evaluated in a phase 1 clinical trial in patients with anemia due to myeloproliferative disorders (NCT04455841).

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

Results of a Phase 1 Trial Testing the Novel Combination Therapy of Venetoclax and Ruxolitinib in Relapsed/Refractory Acute Myeloid Leukemia Patients

Introduction : A functional small molecule screen on a large cohort of primary AML patient samples revealed that the combination of Ruxolitinib (Rux) and Venetoclax (Ven) exhibited ex vivo efficacy and synergy in both newly diagnosed and R/R AML. This observation motivated a Phase I multi-center trial to evaluate Rux+Ven in R/R AML, for which we now report complete toxicity and efficacy data for 30 subjects. Methods: This study was designed to identify a maximum tolerated or recommended Phase II dose (RP2D) and evaluate the overall safety and preliminary efficacy of Rux+Ven therapy. Eligible subjects were ≥18 years old with R/R AML. There was no restriction on the number of previous therapies or prior exposure to Ven or Rux. We included subjects with prior allogeneic transplant if GVHD was controlled and prior MDS (≥75 years old or having poor cardiac or kidney function) who progressed on a hypomethylating agent (HMA) but had no primary AML therapy. In cohorts of 3 to 4, subjects were assigned to receive Rux+Ven at 1 of 6 dose levels according to the “keyboard” Bayesian toxicity probability interval design applied to a target dose-limiting toxicity (DLT) rate of 30%. DLTs were defined as non-disease-related, Grade ≥3 non-hematologic toxicities, with exceptions for nausea, vomiting, infections, febrile neutropenia episodes, and electrolyte abnormalities that resolved within 48 hours. Hematologic DLT was specified as Grade 4 neutropenia after 42 days in the absence of disease. The DLT evaluation period covered the first cycle. Subjects could continue study therapy after two 28-day cycles if they had a response (ie, CR/CRi or MLFS) or were otherwise deriving clinical benefit. Results : This Phase 1 study has completed enrollment (n=30), with all but one patient off-treatment as of June 2023. Baseline patient characteristics are summarized in Table 1. Median age was 69 (range 29-84), 40% of subjects had ≥3 prior therapies, and 43% had prior Ven. At enrollment, 30% had prior MDS, 24% had mutated TP53 (of 25 patients with available data), and 47% had CD56+ blasts. The numbers of patients treated by dose level (DL) were: 3 on DL0 (200 mg qd Ven; 10 mg bid Rux), 4 on DL1 (400 mg qd Ven; 10 mg bid Rux), 3 on DL2 (400 mg qd Ven; 20 mg bid Rux), and 20 on DL3 (400 mg qd Ven; 30 mg bid Rux), the highest dose level of the study. Per prescribing guidelines, the cycle 1 Rux doses of 15 subjects and Ven doses of 20 subjects were reduced because of a concomitant CYP3A inhibitor. The regimen was well-tolerated. As no DLTs were observed, DL3 is the RP2D. The median (interquartile range) duration of therapy was 55 (36-93) days. The most common grade ≥3 hematologic AEs, regardless of attribution, were thrombocytopenia (50%; which was 30% at study enrollment), anemia (40%; 40% at enrollment), neutropenia (37%; 10% at enrollment), febrile neutropenia (33%), and leukopenia (33%; 7% at enrollment). Six subjects had a Grade 5 AE: 4 were unrelated to study drug (2 death NOS, 1 lung infection, 1 respiratory failure) and 2 were potentially related to Ven (1 sepsis, 1 neutropenic pneumonia). Over the first 2 cycles of study therapy, the Clinical Benefit Rate (CBR; defined as the proportion of subjects obtaining at least stable disease) was 63% (95% CI: 44%-80%). Morphologic leukemia-free state (MLFS) was achieved in 20% (95% CI: 8%-39%) and Composite Complete Remission in 10% (95% CI: 2%-27%). Median overall survival (OS) was 3.7 months (95% CI: 2.3-6.5), with 23% (95% CI: 10%-39%) alive 1 year after commencing Rux+Ven. Two participants were exceptional responders: the first achieving MLFS and receiving 16 cycles of study therapy and the second having a complete remission and remaining on-treatment after 31 cycles. Pre-Rux+Ven CD56 expression on blasts correlated with worse response (all 6 ≥MLFS patients were CD56-; Fig 1) and overall survival (HR=2.37, p=0.044; Table 1). Correlative studies such as ex vivo drug assessment, genomic analysis, CyTOF, and single-cell imaging will be presented to enhance this finding. Conclusion: The novel, all-oral combination of Rux+Ven in R/R AML patients was well-tolerated with no DLTs and an encouraging 63% CBR over cycles 1-2. Negative CD56 was a biomarker of response, although additional work is required to determine if this is a broad association with Ven-based therapies or unique to the Rux+Ven combination. The tolerable toxicity profile of Rux+Ven makes it a promising combination to test with HMAs in high-risk AML patients.

Activity of Selinexor As a Single Agent and Synergistic Activity with Approved/Investigational Myelofibrosis Therapies in Vitro

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) that commonly harbors acquired somatic gene mutations in JAK2, CALR or MPL leading to constitutive JAK/STAT activation . Despite the current standard of care, the JAK1/2 inhibitor (JAKi) ruxolitinib (RUX), a significant unmet need remains for patients (pts) with MF, due to lack of response or JAKi resistance. Selinexor (SEL) is an investigational, oral XPO1 inhibitor that may inhibit multiple pathways relevant in MF including JAK/STAT, ERK, and AKT. Previously, we reported that 60 mg SEL in combination with RUX in pts with JAKi-naïve MF resulted in rapid, deep, and sustained spleen responses even in pts receiving suboptimal RUX doses, suggesting synergy between SEL and RUX while maintaining a generally manageable safety profile (Ali et al., ASCO 2023; Ali et al., AACR 2023). Here, we investigated the combinatorial effects of SEL with 5 approved/investigational MF therapeutic agents on human cell lines, then evaluated relevant downstream pathways impacted by SEL +/- RUX. Methods: JAK2 V617F mutant cell lines HEL ( TP53M113K), UKE-1 ( TP53WT), MUTZ-8 ( TP53WT), and JAK2WT ELF-153 ( TP53I251N) cells were treated with SEL alone or in combination with RUX, pacritinib (PAC), momelotinib (MMB), pelabresib (PELA), or navitoclax (NAV). Compounds were added to 1x10 4 cells in a 1:3 or 1:2 serial dilution, then cell viability was assessed 72 hours post-exposure. Single-agent non-linear regressions were used to determine inhibitory concentrations (IC), and drug synergy was evaluated using Synergy Finder 3.0 (Bliss). HEL ( JAK2V617F) and ELF-153 ( JAK2wt) cells were used for mechanistic studies including western blots and RNA sequencing. Results: Single agent SEL reduced cell viability in all cell lines tested, independent of JAK2 mutation status: HEL (IC 50 320nM), UKE-1 (320 nM), MUTZ-8 (12nM) and ELF-153 (570 nM) cells. SEL antiproliferative activity was more potent than RUX (IC 50 range 1-64.6 µM), MMB (1.9-27.8 µM), NAV (0.165-51.4 µM) and PELA (7.5-65.9 µM). When determined by Bliss scores, evidence of synergy was observed in the following co-treatments: SEL + RUX in HEL (10.8) and UKE-1 (11.9) cells; SEL + PELA in HEL (9.9) and UKE-1 (7.7); SEL + MMB (16.1) and NAV (12.4) in HEL ( Figure 1). Additive effects were observed: SEL + RUX in MUTZ-8 and ELF-153 ( JAK2wt) cells; SEL + MMB in UKE-1, MUTZ-8 ELF-153 cells; SEL + PAC in HEL and ELF-153 cells; SEL+ PELA in UKE-1, MUTZ-8 and ELF-153 cells; SEL+NAV in UKE-1 and ELF-153 cells. Mechanistically, SEL and the SEL + RUX combo both induced XPO1 protein degradation to a similar extent. The SEL + RUX combination in HEL cells resulted in a pro-apoptotic effect via decreased MCL-1 protein (P≤0.0001) and increased cleaved PARP vs RUX treatment. Preliminary analysis of mRNA transcript alterations after SEL + RUX treatment showed a modulation of expression of genes related to proliferation ( MYC), senescence ( CDKN1A/B) and cell cycle ( CYCLIND1/A2/B1). A RUX-resistant HEL cell clone was generated via culture with increasing RUX, resulting in a &amp;gt;10-fold increase in RUX IC 50. The SEL IC 50 of the resistant clone was not changed compared to parental cells. RUX-resistant HEL cells showed downregulation of several pathways including MYC, G2M checkpoint, ribosomal biogenesis, proteasome, spliceosome, and nuclear export when compared to parental HEL cells. SEL had synergistic or additive effects with MF agents in this resistant subclone. Conclusion: In a panel of MPN-derived cells with or without JAK2V617F, SEL showed single-agent antiproliferative activity, and synergism with other MF therapeutics at clinically achievable concentrations through regulation of XPO1, JAK/STAT signaling, and apoptosis/senescence regulators. In addition to positive Phase 1 clinical data in pts with JAKi-naïve MF who received SEL with RUX, these preclinical data support synergistic activity between SEL and other MF therapies, suggesting the potential for SEL as a backbone for novel treatment combinations for MF.

Ruxolitinib accelerates influenza A virus adaptation in the Madin-Darby canine kidney (MDCK) cell line.

To investigate the effect of ruxolitinib medium supplement, separately and in combination with trypsin, on influenza A virus (IAV) adaptation and propagation in the Madin-Darby canine kidney (MDCK) cell line. Two consecutive passages of three egg-based IAV strains were performed in the MDCK cell line with medium (a) without additives; (b) with a combination of ruxolitinib and trypsin; (c) with ruxolitinib; and (d) trypsin. Adaptation without a medium additive failed in both passages. After a single passage, the probability of the IAV adaptation was highly significantly influenced by the type of additive (binomial generalized linear model, χ22=23.84, P<0.00001). The highest probability of adaptation was achieved with the combination of ruxolitinib and trypsin, followed by ruxolitinib alone and trypsin. After the two consecutive passages, the influence of the type of medium additive on the probability of virus adaptation was no longer significant. In two of three IAV MDCK-adapted strains, the type of medium additive had no significant influence on virus yields. Ruxolitinib accelerates the adaptation of IAV in the MDCK cell line both individually and together with trypsin.

Potential drug repurposing of ruxolitinib to inhibit the JAK/STAT pathway for the treatment of patients with epithelial ovarian cancer.

This review aimed to describe the potential for therapeutic targeting of the JAK/STAT signaling pathway by repurposing the clinically-approved JAK inhibitor ruxolitinib in the patients with epithelial ovarian cancer (OC) setting. We reviewed publications that focus on the inhibition of the JAK/STAT pathway in hematological and solid malignancies including OC. Preclinical studies showed that ruxolitinib effectively reduces OC cell viability and metastasis and enhances the anti-tumor activity of chemotherapy drugs. There are a number of recent clinical trials exploring the role of JAK/STAT inhibition in solid cancers including OC. Early results have not adequately supported efficacy in solid tumors. However, there are preclinical data and clinical studies supporting the use of ruxolitinib in combination with both chemotherapy and other targeted drugs in OC setting. Inflammatory conditions and persistent activation of the JAK/STAT pathway are associated with tumourigenesis and chemoresistance, and therapeutic blockade of this pathway shows promising results. For women with OC, clinical investigation exploring the role of ruxolitinib in combination with chemotherapy agents or other targeted therapeutics is warranted.

Effect of ruxolitinib and magrolimab on calreticulin in myelofibrosis CD34+ cells in vitro: Proof of concept for combination therapy.

118 Background: In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. We have previously found that CD47, not CALR, is overexpressed on the membrane of patients with MPN. Anti-CD47 Magrolimab, is currently being evaluated in clinical trials in AML, MDS but have yet to be tested, either in monotherapy or in combination in MF. Methods: Mononuclear cells were collected by Ficoll separation from bone marrow of 6 MF patients and from 2 controls. CD34+ cells and monocytes were purified using Dynabeads kits from Thermofisher. Cells expression of CALR and CD47 was measured by flow cytometry, before and after incubation with ruxolitinib (Jak inhibitor) alone and in combination with magrolimab. Cells survival was calculated after combining CD34 with monocytes. Informed Consent was obtained for all samples collected. Results: CD47 membrane expression was 57% in untreated MF cells compared to 12% in the control samples and CALR membrane expression was 51.8% in untreated MF cell compared to 12.5% in the control samples. After incubation with ruxolitinib, MF CD34+ cell survival decreased to 98.2%. Following Incubation with magrolimab alone, MF CD34+ cells show no changes in survival (100% alive). The combination of ruxolitinib and magrolimab reduced slightly the MF CD34+ cell survival to 92%. A substantial membrane overexpression of CALR was observed in MF CD34+ cells when incubated with Ruxo and Magrolimab, 74.1%, vs Ruxo alone 52.6%, Magrolimab alone 53.4% and in untreated (51.8%). Interestingly, in the healthy controls, the exposure to combination therapy did not cause the same degree of CALR overexpression (20.7% vs 12.5%), indicating a much stronger pro-phagocytic effect following magrolimab/ruxolitinib combo in MF cancer cells. Conclusions: In vitro, treatment with ruxolitinib or ruxolitinib plus magrolimab, slightly reduce the overall cell survival rate in CD34+ MF samples, comparing with no significant changes in single agent magrolimab. However, the combination of Ruxolitinib and Magrolimab significantly increases CALR membrane expression comparing with Ruxo or Magrolimab alone, signalling a much stronger pro-phagocytic message in MF cells comparing with controls. The induced CALR over-expression, could stimulate a stronger immune response in vivo, where the presence of macrophages would allow removal of CALR rich cells and potentiate the cytoreductive activity towards MF clone. These data support the rational of combining Ruxolitinib with Magrolimab in the next clinical trial stage in myelofibrosis.

Effects of ruxolitinib and magrolimab on calreticulin in myelofibrosis CD34+ cells in vitro: Proof of concept for combination therapy.

7064 Background: In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. We have previously found that CD47, not CALR, is overexpressed on the membrane of patients with MPN. Anti-CD47 Magrolimab, is currently being evaluated in clinical trials in AML, MDS but have yet to be tested, either in monotherapy or in combination in MF. Methods: Mononuclear cells were collected by Ficoll separation from bone marrow of 6 MF patients and from 2 controls. CD34+ cells and monocytes were purified using Dynabeads kits from Thermofisher. Cells expression of CALR and CD47 was measured by flow cytometry, before and after incubation with ruxolitinib (Jak inhibitor) alone and in combination with magrolimab. Cells survival was calculated after combining CD34 with monocytes. Informed Consent was obtained for all samples collected. Results: CD47 membrane expression was 57% in untreated MF cells compared to 12% in the control samples and CALR membrane expression was 51.8% in untreated MF cell compared to 12.5% in the control samples. After incubation with ruxolitinib, MF CD34+ cell survival decreased to 98.2%. Following Incubation with magrolimab alone, MF CD34+ cells show no changes in survival (100% alive). The combination of ruxolitinib and magrolimab reduced slightly the MF CD34+ cell survival to 92%. A substantial membrane overexpression of CALR was observed in MF CD34+ cells when incubated with Ruxo and Magrolimab, 74.1%, vs Ruxo alone 52.6%, Magrolimab alone 53.4% and in untreated (51.8%). Interestingly, in the healthy controls, the exposure to combination therapy did not cause the same degree of CALR overexpression (20.7% vs 12.5%), indicating a much stronger pro-phagocytic effect following magrolimab/ruxolitinib combo in MF cancer cells. Conclusions: In vitro, treatment with ruxolitinib or ruxolitinib plus magrolimab, slightly reduce the overall cell survival rate in CD34+ MF samples, comparing with no significant changes in single agent magrolimab. However, the combination of Ruxolitinib and Magrolimab significantly increases CALR membrane expression comparing with Ruxo or Magrolimab alone, signalling a much stronger pro-phagocytic message in MF cells comparing with controls. The induced CALR over-expression, could stimulate a stronger immune response in vivo, where the presence of macrophages would allow removal of CALR rich cells and potentiate the cytoreductive activity towards MF clone. These data support the rational of combining Ruxolitinib with Magrolimab in the next clinical trial stage in myelofibrosis.

A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis.

TPS7086 Background: Patients (pts) with myelofibrosis (MF) have a reduced life-expectancy compared to age-matched controls. Several oral Janus Kinase (JAK) inhibitors such as ruxolitinib (RUX) have been approved by the US FDA based on improvements in symptom burden and spleen volume reduction compared to physician’s choice of best available therapy. However, duration of response to these agents is generally time-limited and treatment with JAK inhibitors is not considered to have disease-modifying potential. Additionally, median overall survival after RUX discontinuation is only 11-14 months. Thus, novel mechanism-based therapies for pts with disease progression on RUX are needed. JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A, which is also upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been implicated in MF pathogenesis. Finally, we and others have shown that combination of the CDK4/6 inhibitors and RUX had synergistic effects in murine models of MF (Rampal et al., CCR 2021) providing the preclinical rationale for this trial. Methods: This multicenter, phase I dose-escalation trial (NCT05714072) evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in pts with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. Among the currently FDA-approved CDK4/6 inhibitors, abemaciclib was chosen for combination therapy due to less myelosuppressive effects compared with other CDK4/6 inhibitors. Inadequate response to RUX is defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms, as defined by the presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS. Following a conventional “3+3” dose-escalation design, pts will be treated with increasing doses of abemaciclib and stable doses of RUX (10mg or 15mg BID). The primary endpoint is to determine the maximum tolerated dose of RUX + abemaciclib. Secondary endpoints include measures of efficacy. Exploratory endpoints include changes in allele fraction of driver mutations, gene expression profiling, and clonal architecture to identify biomarkers of response, mechanisms of resistance, and to assess for any disease-modifying effects of the combination therapy. This trial is open to enrollment at Memorial Sloan Kettering Cancer Center and MD Anderson Cancer Center. Clinical trial information: NCT05714072 .

Safety and feasibility of JAK inhibitor ruxolitinib in newly-diagnosed high-grade gliomas (CRUX): Final toxicity report.

2060 Background: 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. This concept was explored in a phase I trial (NCT03514069), whose final report on toxicity is presented here. 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. The primary study objective was the determination of the maximum tolerated dose (MTD) of ruxolitinib in combination with chemoradiation. The secondary objective was the determination of safety, overall survival (OS), and progression-free survival (PFS). The exploratory aims were to investigate relationships between clinical outcomes and genomic signatures. Results: 60 patients were enrolled, with a median age of 60.5 years (range 22-78). 23 (38%) patients were female and 37 (62%) were male. 29 (48%) were MGMT unmethylated and received ruxolitinib with radiation of 60 Gy over 6 weeks. 31 (52%) patients were MGMT methylated and received ruxolitinib with 75 mg/m2 of temozolomide (TMZ) with radiation of 60 Gy over 6 weeks. The 1-year OS rate was 77% for all GBM patients; 62% for arm 1 (unmethylated MGMT) and 93% for arm 2 (methylated MGMT). Median OS for arm 1 was 18.1 months (10.1, NA) and was not reached for arm 2. 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. Conclusions: Ruxolitinib therapy is safe and feasible in combination with TMZ and radiation. Efficacy of ruxolitinib plus SOC appears promising compared to historical benchmarks for both MGMT methylated and MGMT unmethylated cohorts. A randomized phase 2 trial is planned. Clinical trial information: NCT03514069 .