FRACTION: protocol of a phase II study of Fedratinib and Nivolumab combination in patients with myelofibrosis and resistance or suboptimal response to JAK-inhibitor treatment of the German MPN study group (GSG-MPN)

Development of Janus-kinase (JAK) inhibitors has revolutionized the therapeutic landscape for patients with myeloproliferative neoplasia (MPN). Following approval of the first JAK1/2-inhibitor Ruxolitinib, symptoms of this inflammatory disease, characterized by splenomegaly, release of inflammatory cytokines and appearance of thrombosis, could be effectively reduced for the first time. However, JAK-inhibitor treatment is limited in several aspects: 1) duration of response: 3 years after initiation of therapy more than 50% of patients have discontinued JAK-inhibitor treatment due to lack of efficacy or resistance; 2) reduction of disease burden: while effective in reducing inflammation and constitutional symptoms, JAK-inhibitors fail to reduce the malignant clone in the majority of patients and therefore lack long-term efficacy. Early clinical trials for patients with myelofibrosis (MF) have tried to address these issues for patients with suboptimal response to Ruxolitinib therapy while combination therapies with Fedratinib are rare. Recent reports provided first evidence on how the JAK2-V617F mutated myeloid cells may influence T-cell responses. JAK2-V617F promoted the synthesis of PD-L1 in MPN cells leading to limited anti-neoplastic T-cell responses, metabolic changes in T-cells and eventually JAK2-V617F-driven immune-escape of MPN cells. These findings may facilitate the use of immunotherapeutic approaches for JAK-mutated clones. Immune checkpoints refer to a variety of inhibitory pathways that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. The FRACTION study is a single arm, open label Phase II trial investigating the combination of Fedratinib with the PD-1 inhibitor Nivolumab in patients with myelofibrosis and suboptimal or lack of response to JAK-inhibitor therapy. Over a 12 months period the trial assesses longer term outcomes, particularly the effects on clinical outcomes, such as induction of clinical remissions, quality of life and improvement of anemia. No prospective clinical trial data exist for combinations of JAK- and immune-checkpoint-inhibitors in the planned MF study population and this study will provide new findings that may contribute to advancing the treatment landscape for MF patients with suboptimal responses and limited alternatives. Supplementary Information The online version contains supplementary material available at 10.1007/s00277-024-05867-w.


Introduction
Development of Janus-kinase inhibitors has revolutionized the therapeutic landscape for patients with myeloproliferative neoplasia (MPN).Following approval of the first Extended author information available on the last page of the article Jak1/2-inhibitor Ruxolitinib (Rux), symptoms of this inflammatory disease, characterized by splenomegaly, release of inflammatory cytokines and appearance of thrombosis, could be effectively reduced for the first time [1].However, JAK-inhibitor treatment is limited in several aspects: (i) duration of response: 3 years after treatment more than 50% of patients have discontinued JAK-inhibitor treatment due to lack of efficacy or resistance [2,3].(ii) reduction of disease burden: while effective in reducing inflammation and constitutional symptoms, JAK-inhibitors fail to reduce the malignant clone in the majority of patients and therefore lacks long-term efficacy [4].
Clinical trials for patients with myelofibrosis (MF) have tried to address these issues for patients with suboptimal response to Ruxolitinib therapy.Combination therapies have not been evaluated with Fedratinib so far.Potential combination partners include signaling inhibitors, epigenetic drugs and immunotherapeutics, among others.
The consequences of JAK-inhibition on human immune cell function have been studied in more detail.In early clinical trials, increased numbers of viral infections have been described in MPN patients on treatment with the JAK1/2 inhibitor Ruxolitinib [5,6].Moreover, JAK1/2-inhibitors such as Ruxolitinib have been used successfully in rheumatological diseases as well as in the setting of allogeneic transplantation as treatment of acute and chronic graftversus-host disease (GvHD).In murine models of GvHD Ruxolitinib enhanced survival and limited proinflammatory cytokine production as well as Th1 and Th17 polarization.In MPN patients, JAK1/2 inhibition leads to reduction in CD3 + T-cells and decreased cytokine production.Within the T-cell compartment regulatory T-cells (Treg) and Th1 cells seem to be most prominently affected [7][8][9].Also, effector functions of CD8 + T-cells are impaired upon JAK1/2 inhibitor treatment [10].Furthermore, JAKinhibition compromises B-cell differentiation and antibody production [11,12] as well as dendritic cell function [13].Therefore, it is crucial to determine whether inhibitors targeting JAK1, JAK2, or both could impair T-cell function.JAK1/2 inhibitors as well as JAK2 specific inhibitors had been studied in vitro and in vivo on healthy donor T-cells.Exposure to either JAK1/2 inhibitor resulted in the inhibition of proliferation, global activation (CD69), and STAT1 phosphorylation of CD4 + and CD8 + T-cells, while selective JAK2 inhibition had no such effect.Likewise, when using genetic inactivation of either JAK1 or JAK2 in T-cells only JAK1 depletion was sufficient to inhibit global T-cell function in vitro.JAK2 was dispensable for global T-cell effector functions in mouse models of GvHD.These findings underscore the importance of JAK-selectivity depending on the underlying condition or context.[14].Consistently, while there were signals for increased rates of infectious complications upon Ruxolitinib treatment in clinical trials, there was no increased incidence in trials using Fedratinib [6,[15][16][17], with a relatively low rate of infectious complications.These findings indicate that JAK2-specific inhibitors like Fedratinib allows immune responses and therefore may serve as a combination partner for immunotherapeutic agents.
Fedratinib is an oral wild type and mutated Janus Kinase 2 (JAK2) and (FLT3) inhibitor.Fedratinib is JAK2-selective with higher potency for JAK2 over family members JAK1, JAK3 and TYK2.In cell models expressing mutationally active JAK2 or FLT3, Fedratinib reduced phosphorylation of STAT3 and 5 proteins, inhibited cell proliferation, and induced apoptosis.In mouse models of JAK2V617F-driven myeloproliferative neoplasms, Fedratinib inhibited phosphorylation of STAT3/5, increased survival and improved MPN-associated symptoms.This included reduction of white blood cells, hematocrit, splenomegaly, and fibrosis.Fedratinib has been studied extensively in the treatment of patients with myelofibrosis and has recently demonstrated clinical efficacy in a randomized, placebo-controlled, Phase 3 study (JAKARTA) in patients with intermediate-2 or highrisk MF [18,19].
Most notably, recent reports provided first evidence on how the JAK2-V617F mutated myeloid cells may influence T-cell responses.JAK2-V617F promoted the synthesis of PD-L1 in MPN cells leading to limited anti-neoplastic T-cell responses, metabolic changes in T-cells and eventually JAK2-V617Fdriven immune-escape of MPN cells [20].These findings may facilitate the use of immunotherapeutic approaches for JAK-mutated clones.Immune checkpoints refer to a variety of inhibitory pathways that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage.Checkpoint inhibitors, such as ipilimumab targeting CTLA-4, pembrolizumab and Nivolumab targeting programmed death-1 (PD-1), have been approved for numerous cancer indications including hematologic malignancies.Nivolumab is one of the most extensively studied immune checkpoint inhibitors across various tumor types.Nivolumab was the first PD-1 immune checkpoint inhibitor to be approved for use in advanced, squamous non-small cell lung cancer (NSCLC) following prior chemotherapy.Nivolumab is a human immunoglobulin G4 (IgG4) monoclonal antibody.It binds to the PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, thus releasing PD-1 pathway-mediated inhibition of the immune response, including the antitumor immune response.
Checkpoint inhibitors Nivolumab and Pembrolizumab have been investigated as monotherapy in patients with myelofibrosis.However, treatment duration was rather short due to the lack of early responses.Of note, immune responses upon checkpoint inhibitor treatment may require a prolonged treatment period to facilitate measurable clinical responses.
Upon monotherapy, changes in the immune milieu could still be recorded, further suggesting that a prolonged treatment may facilitate improved responses.Therefore, Fedratinib was a logical combination partner for checkpoint inhibitors as it is an effective JAK-inhibitor to reduce spleen size and symptoms while allowing immune responses over a prolonged period of treatment.
The aim of this phase 2 trial is therefore to evaluate the clinical efficacy of Fedratinib and Nivolumab combination therapy in patients with primary and secondary MF based on the consensus criteria of the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT), extended by the criterion RBC-transfusion independence (RBC-TI).

Study design and objectives
This study is an open-label, single-arm, phase-II trial to assess the efficacy of a drug combination of Fedratinib and Nivolumab in primary (pre-fibrotic or overt) and secondary MF patients with no response or suboptimal response to any JAK-inhibitor therapy (regarding persistence of symptoms, splenomegaly, cytopenia or hyperproliferation) OR failure [secondary resistance] to JAK-inhibitor treatment as defined by IWG-MRT criteria.Dosing regimen of the drugs is derived from previous studies with either Fedratinib or Nivolumab.Fedratinib treatment will be started at 400 mg QD, whereas the dose of Nivolumab will be started in cycle 2 at a fixed dose of 240 mg i.v.every 2 weeks (Fig. 1).Dose adjustment or discontinuation is intended for both drugs in case of severe hematologic and/or non-hematologic toxicity.Treatment cycles are defined as 28 days.The estimated total duration of the study for each patient is 12 months.Patients whose disease responds or who show at least a clinical benefit may continue to receive the combination.Patients will receive study treatment until progressive disease or relapse (acc.to IWG-MRT), death or study discontinuation for other reasons.Both drugs do not have an overlapping toxicity profile as they target distinct biological pathways (JAK2inhibitor; immune-checkpoint inhibitor).Both classes of compounds have been investigated in phase 2 and 3 trials without excess of toxicity [21][22][23][24][25][26][27].
Primary objective is the evaluation of the clinical efficacy of Fedratinib and Nivolumab combination therapy in primary and secondary MF patients based on the consensus criteria of the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) [28], extended by the criterion RBC-transfusion independence (RBC-TI) [29,30].Secondary objectives include evaluation of the safety of a combination therapy (Fedratinib and Nivolumab) in patients with primary and secondary MF, of the clinical benefit (defined as prolongation of RBC transfusion intervals by ≥ 50% compared to baseline in transfusion dependent patients or ≥ 1 g/dL Hb increase in the absence of RBC transfusion dependency and/or improvement of at least one MF-associated symptom according by at least 50% and/or improvement of ≥ 2 MF-associated symptoms by at least 25%), progression-free survival, response duration, disease burden (allelic ratio of the respective driver mutation and of high-risk mutations by next-generation sequencing [NGS]), fibrosis grade and overall survival, the quality of

Study population
The inclusion and exclusion criteria for this study are outlined in Table 1.Major eligibility criteria include adult female and male patients ≥ 18 years of age diagnosed with myelofibrosis (MF) according to the WHO 2008 or 2016 criteria, including primary (pre-fibrotic or overt) and secondary myelofibrosis.The study focuses on patients with an indication for therapy (either symptomatic patients with splenomegaly > 11 cm diameter and/or symptoms restricting their daily activity or patients with DIPSS int-2, or high risk or MIPSS70 int or high).Patients with failure [secondary resistance] to JAK-inhibitor treatment as defined by IWG-MRT criteria can be enrolled as well as patients with no response or suboptimal response to any JAK-inhibitor therapy (regarding persistence of symptoms, splenomegaly, cytopenia or hyperproliferation) defined either by persisting splenomegaly (> 11 cm total diameter), persisting leukoerythroblastosis, anemia < 6.2 mmol/l (< 10 g/dl), elevated WBC (> 11 Gpt/l) or persisting general or constitutional symptoms (persistence is defined as less than 50% reduction to baseline when using the MPN10 TSS Score).

Study drugs
Fedratinib capsules will be administered orally, QD, 400 mg (100 mg capsules will be provided: 4 capsules of 100 mg per day).Fedratinib treatment will be administered as a run-in phase for 4 weeks.Fedratinib dose modifications will be allowed based on observed toxicity to a 300 mg or 200 mg, or 100 mg daily dose or may be temporarily discontinued in this study.For the first 2 cycles, a fixed dose of 400 mg should be maintained with appropriate supportive medication, as major toxicities have been shown to decrease with increasing number of treatment cycles [31].Provisions are in place to allow further dose reduction for subjects with comedication with moderate or strong Cytochrome P4503A4 (CYP3A4) inhibitors.
From cycle 2 on, Nivolumab will be added at a fixed dose of 240 mg i.v., every 2 weeks.The frequency of Nivolumab infusion can be reduced based on observed toxicity.Infusions can be continued even if Fedratinib is temporarily discontinued.

Study assessments
Treatment response will be evaluated continuously after each treatment cycle (1 cycle = 28 days) according to the IWG-MRT criteria expanded by the response criterion of red cell transfusion (RCT)-independency.In case of progressive disease, study treatment will be stopped; in patients showing response or stable disease with or without clinical benefit (defined as prolongation of RBC transfusion intervals by ≥ 50% compared to baseline in transfusion dependent patients or ≥ 1 g/dL Hb increase in the absence of RBC transfusion dependency and/or improvement of at least one MF-associated symptom according by a minimum of 50% and/or improvement of ≥ 2 MF-associated symptoms by a minimum of 25%) treatment continuation is intended until disease progression or other reasons for withdrawal.Conditions leading to patient withdrawal from the study are detailed in Supplementary Fig. 1.All patients should be screened for inclusion and exclusion criteria within 28 days prior to the first dose of Fedratinib (screening-phase).Detailed study procedures can be found in Table 2.

Outcomes
The primary efficacy endpoint of the study will assess the best response rate within 12 treatment cycles according to the IWG-MRT criteria (including complete remission, CR, partial remission, PR, clinical improvement CI, stable disease, SD) [28], and RCT independency according to Gale et al. [29,30].
Secondary endpoints include the overall safety profile of Fedratinib and Nivolumab combination characterized by the type, frequency, severity (graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE]), timing and relatedness of adverse events (AEs) and laboratory abnormalities observed during treatment, as well as the cumulative incidence of leukemic transformation, clinical benefit, progression-free survival (PFS), duration of response, overall survival (OS) and reduction of disease burden (Table 3).
Exploratory endpoints include: Change in ECOG performance status from study entry to each visit at which the variable is measured, investigation of immune-cell expansion and immune-cell responses to checkpointinhibitor therapy at baseline (before first IMP dosing), after 6 months study treatment, and after 12 months study treatment (or at end of treatment (EOT)), assessment of disease burden measured as allelic burden of the respective driver mutations (JAK2, CALR, MPL) at baseline (before first investigational medical product (IMP) dosing), after 6 months study treatment and after 12 months study treatment (or at EOT), assessment of clonal diversity and evolution by NGS-sequencing of a defined myeloid gene panel at baseline (before first IMP dosing), after 6 months study treatment and after 12 months study treatment (or at EOT) as well as assessment of bone marrow fibrosis by central histology at baseline before first IMP dosing (screening period) and after 12 months study treatment (or Patients with an indication for therapy (either symptomatic patients with splenomegaly > 11 cm diameter and/or symptoms restricting their daily activity or patients with DIPSS int-2, or high risk or MIPSS70 int or high) e. Patients with no response or suboptimal response to any JAK-inhibitor therapy (regarding persistence of symptoms, splenomegaly, cytopenia or hyperproliferation) defined either by ANY ONE of the following criteria (f-k): f.Persisting Splenomegaly > 11 cm total diameter g.Persisting leukoerythroblastosis h.Anemia < 6.2 mmol/l (< 10 g/dl) i. Elevated WBC (> 11 Gpt/l) j.Persisting general or constitutional symptoms (persistence is defined as less than 50% reduction to baseline when using the MPN10 TSS Score) OR k. failure [secondary resistance] to JAK-inhibitor treatment as defined by IWG-MRT criteria l.ECOG performance status < 3 at screening and adequate organ function m.Reliable contraception should be maintained throughout the study and for 1 month after discontinuation of Fedratinib or 5 months after discontinuation of Nivolumab** n.Subject must be willing to receive transfusion of blood products o.Thiamine levels not below lower limit of normal (prior substitution is possible) p.Normal nutritional status, as judged by the physician q.Females of childbearing potential (FCBP) must undergo repetitive pregnancy testing (serum or urine) and pregnancy results must be negative r. Unless practicing complete abstinence from heterosexual intercourse, sexually active FCBP must agree to use adequate contraceptive methods (i.e.failure rate of < 1% per year) s.Males (including those who have had a vasectomy) must use barrier contraception (condoms) when engaging in sexual activity with FCBP.
Males must agree not to donate semen or sperm *There are no data that indicate special gender distribution and the risk to be diagnosed with myelofibrosis (MF) does not depend on a patient's gender.Therefore, patients will be enrolled in the study gender-independently

Exclusion Criteria
The presence of ANY of the following criteria will exclude a patient from study enrollment: a. Planned hematopoietic stem cell transplantation within 3 months and suitable donor available b. > 10% blasts in bone marrow smear (cytology) or > 2 × in blood smear within the screening phase or > 20% blasts at any time in bone marrow or peripheral blood smears c.Creatinine > 2xN and Creatinine-Clearance < 45 ml/min; ALAT, ASAT & bilirubin > 3xN (if MF impact on liver > 5xN) d.Baseline platelets count below 50 × 10 9 /L and ANC < 1.0 × 10 9 /L e. Diagnosis of PV, ET (according to WHO 2016) or positive molecular test for BCR-ABL f.Patients on ongoing medication for myelofibrosis including systemic corticosteroids (detailed list of permitted medications is provided in paragraph 9.1.10.4 and Appendix V).Use of steroids within 14 days prior to the first dose of study drug and until end of treatment is prohibited by patients g.Uncontrolled infection h.Evidence of acute or chronic infection with hepatitis B, hepatitis C, human immunodeficiency virus (HIV) or tuberculosis i.Current participation in any other interventional clinical study within 30 days before the first administration of the investigational product or at any time during the study, unless it is an observational (non-interventional) study, or during the follow-up period of an interventional study with last dose of investigational product ≥ 30 days prior first administration of investigational product within this study j.No consent for registration, storage and processing of the individual disease characteristics and course as well as information of the family physician about study participation k.No consent for biobanking of patient's biological specimens l.Prior therapy with checkpoint-inhibitors m.Vaccination within 4 weeks prior to treatment start n.Hypersensitivity to the IMPs or to any of the excipients o.History of or uncontrolled autoimmune disease such as autoimmune-hepatitis, -pneumonitis, -thyroiditis, chronic inflammatory bowel disease, multiple sclerosis, or rheumatologic diseases (including but not limited to systemic lupus and vasculitis) p. History of malignancy except for i) adequately treated local basal cell or squamous cell carcinoma of the skin, ii) asymptomatic prostate cancer without known metastatic disease and with no requirement for therapy or requiring only hormonal therapy and with normal prostate-specific antigen for ≥ 1 year prior to randomization, or iii) any other cancer that has been in complete remission for ≥ 5 years q.Secondary malignancy that limits survival to less than 6 months r.Drug or alcohol abuse within the last 6 months s.Patients who cannot adhere to the Pregnancy Prevention Plan t.Pregnant or breast-feeding females u.Thiamine levels below normal limit despite supplementation v. Patients who are unable to consent because they do not understand the nature, significance and implications of the clinical trial and therefore cannot form a rational intention in the light of the facts [ § 40 Abs.Safety endpoints include hematologic (thrombocytopenia, anemia, and neutropenia) and non-hematological toxicities.All adverse events / toxicities, including serious adverse events, are graded according to NCI CTCAE Version 5.0.Safety assessments will consist of evaluating adverse events, laboratory parameters including hematology and chemistry, vital signs, and physical examinations.Safety will be ensured by internal and external supervision.

Statistical analyses and sample size
The sample size calculation is based on the A'Hern's singlestage design [32].The assumptions used for statistical power calculations are that the true target response rate of 20% (alternative hypothesis) will be tested against the null hypothesis response rate of 5%, with a type I error rate of 0.05 and a power of 80%.A response is defined as achieving a response according to the IWG-MRT criteria.If the number of patients with a response is 4 or more, the null hypothesis is rejected with an actual error rate of alpha < 0.05.If the number of responses is 3 or less, the alternative hypothesis (P ≥ 0.2) is rejected with an actual Table 3 Endpoints Primary Endpoint Best response rate within 12 treatment cycles according to the IWG-MRT criteria (including complete remission, CR, partial remission, PR, clinical improvement, CI, stable disease, SD [28], and red cell transfusion (RCT) independency according to Gale et al. [29,30]) Secondary Endpoints • Overall safety profile of Fedratinib and Nivolumab combination characterized by type, frequency, severity (graded using the National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE]), timing and relatedness of adverse events (AEs) and laboratory abnormalities observed during treatment, as well as cumulative incidence of leukemic transformation • clinical benefit • progression-free survival, • duration of response, • overall survival • reduction of disease burden Exploratory Endpoints • Quality of life assessed by the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF, MPN10), change in ECOG performance status from study entry to each visit where the variable is measured • Adherence assessed by modified Morisky 8-item questionnaire • Investigation of immune-cell expansion and immune-cell responses to checkpoint-inhibitor therapy at baseline (before first IMP dosing), after 6 months study treatment, and after 12 months study treatment (or at EOT) • Assessment of disease burden measured as allelic burden of the respective driver mutations (JAK2, CALR, MPL) at baseline (before first IMP dosing), after 6 months study treatment and after 12 months study treatment (or at EOT) • Assessment of clonal diversity and evolution by NGS-sequencing of a defined 32 gene panel at baseline (before first IMP dosing), after 6 months study treatment and after 12 months study treatment (or at EOT) • Assessment of bone marrow fibrosis by central histology at baseline before first IMP dosing (screening period) and after 12 months study treatment (or at EOT) error rate of beta = 0.182 A total of 27 subjects will be needed based on this calculation.A total of 30 patients will need to be recruited in this phase 2 trial assuming an early dropout rate of 10% (n = 3).A formal interim analysis will not be performed.The study is conducted at 9 academic centers in Germany (Supplementary Table S1).

Discussion
Myelofibrosis is a malignant stem-cell disease which is associated with a poor outcome.Overall survival ranges from 5 to 150 months depending on risk factors present at diagnosis [33].Allogeneic stem cell transplantation remains the only potentially curative treatment option.However, treatmentassociated complications are frequent and many patients are not eligible for a transplantation regimen due to age and poor performance status.For these patients, hydroxyurea, androgens, erythropoietin, splenectomy, or splenic radiation are palliative therapeutic options [34].None of these therapies have been proven to prolong survival in MF, and cytopenia in particular remains an unsolved problem.
The discovery of the Janus kinase (JAK) pathway's role in MF pathogenesis has led to the development of targeted therapies, notably JAK inhibitors.Currently, approval and clinical application of 4 JAK inhibitors (Ruxolitinib, Fedratinib, Momelotinib, and Pacritinib) is established in the treatment of MF.Ruxolitinib, the first JAK inhibitor approved for MF, targets JAK1 and JAK2 pathways.It has been shown to significantly reduce splenomegaly and alleviate symptoms in MF patients, improving overall survival compared to conventional therapies.However, its use is limited by cytopenias, particularly thrombocytopenia and anemia.Momelotinib targets JAK1, JAK2, and ACVR1, thereby addressing both splenomegaly and anemia, a common complication in MF.It stands out for its dual efficacy in improving blood counts while controlling disease symptoms, presenting a valuable option for patients with anemia.Pacritinib is unique among JAK inhibitors for its minimal myelosuppression.It is specifically indicated for patients with severe thrombocytopenia.Pacritinib targets JAK1, JAK2, FLT3, and IRAK1, and has shown efficacy in reducing spleen size and improving symptoms without exacerbating cytopenias.
Fedratinib, a selective JAK2 inhibitor, has been approved for patients with intermediate-2 or high-risk MF, including those previously treated with Ruxolitinib.It offers a beneficial effect on spleen volume and symptom relief.Fedratinib is distinct for its ability to manage patients with a baseline thrombocytopenia, but it requires monitoring of thiamine levels due to the risk of Wernicke's encephalopathy.Fedratinib is an oral small molecule kinase inhibitor with activity against wild type and mutationally activated JAK2 and FLT3 was granted approval for the treatment of myelofibrosis.Fedratinib is a JAK2-selective inhibitor with higher inhibitory activity for JAK2 over family members JAK1, JAK3, and tyrosine kinase 2 (TYK2).Fedratinib demonstrated potent kinase inhibitory activity against JAK2, JAK2V617F and FLT3, with IC50 values of 3 nM, 3 nM and 15 nM, respectively.In contrast, the selectivity for the closely related JAK1, TYK2 and JAK3 is 35-, 135-and > 300-fold lower, respectively, than for JAK2.In contrast to the severe impairment of T-cell function mediated by Ruxolitinib, which has been (in part) attributed to its inhibitory activity on JAK1 [14] and has facilitated its use in diseases with T-cell hyperreactivity such as Graft-versus-Host Disease (GvHD) [35,36], Fedratinib, does not impair T-and NK-cell responses to a comparable extent [37].This unique profile of Fedratinib in regard to immune responses facilitates a superior profile regarding immunocompetence of MPN patients in clinical use and the option to combine an effective JAK-inhibitor with immunotherapy.
Besides different JAK-inhibitors with different specificity for JAK1 and JAK2, epigenetic modulators, inhibitors of cell signaling, anti-inflammatory compounds and immunotherapies are within focus of interest in MF.Preclinical assessment of checkpoint-inhibitors has shown promising results [20].
Recent phase 2 trials have investigated the use of immune checkpoint-inhibitors in the treatment of myelofibrosis [38,39] (Table 4).Nivolumab as a single-agent therapy has been investigated in a total of 8 patients.While no severe immune-reactions or adverse events were observed, there was also lack of efficacy beyond stable disease.Notably, only 1/8 patients had a prolonged treatment period of more than 6 months, all other patients had discontinued early due to lack of a meaningful response [38].In a second trial, Pembrolizumab was investigated as a single-agent therapy in Ruxolitinib pre-treated patients with myelofibrosis [39].The study followed a Simon 2-stage design and enrolled a total of 10 patients.Pembrolizumab treatment was well tolerated, however, no relevant clinical responses could be recorded, resulting in discontinuation of the trial after the first stage was completed.Immune profiling by flow cytometry, T-cell receptor sequencing, and plasma proteomics demonstrated changes in the immune milieu of patients, which suggested improved T-cell responses that can potentially favor antitumor immunity [39].At 6 months, only 5/9 chronic phase MF patients were still on treatment.
The fact that patients had no relevant responses regarding spleen size and symptom burden may have contributed to their early discontinuation and drop out (Supplementary Table S2).The combination of checkpoint-inhibitors with the first available JAK-inhibitor Ruxolitinib is not possible due to its efficacy on JAK1 leading to impairment of T-cell function.However, Fedratinib, a JAK2-specific compound, has shown to allow immune responses in pre-clinical and clinical settings.This enables a therapeutic window to explore the induction of immune responses by the established checkpoint-inhibitor Nivolumab in combination with the selective JAK2-inhibitor Fedratinib.Checkpoint-inhibitors mediate indirect anti-tumor activity by activating T-cell responses.While Nivolumab can eradicate malignant MPN cells through activation of T-cell responses, Fedratinib predominantly reduces the spleen size and improves constitutional symptoms.As both aspects of disease control are highly relevant and addressing clinical needs in MF, the combined use of the drugs provides a promising therapeutic concept.Therefore, the risk-benefit ratio is based on arguments discussed above and clearly favors to the beneficial side.
The sequential administration of both drugs is poised to mitigate adverse effects: the initiation of Fedratinib dosing at baseline affords patients the opportunity to acclimate to the treatment and mitigate potential gastrointestinal (GI) toxicity.Notably, GI toxicity is the most common adverse event observed with Fedratinib treatment, as elucidated by England and colleagues [40].However, its incidence can be significantly attenuated with concurrent interventions such as anti-emetic prophylaxis or standby anti-diarrhea medication like loperamide.Remarkably, while diarrhea manifested in up to 80% of patients during the early Fedratinib trials, the implementation of supportive medication regimens in the FREEDOM trials substantially reduced these side effects to below 40% [40].
Significant discourse revolves around the definition of meaningful endpoints in myelofibrosis trials.The primary endpoint of the FRACTION trial will be the optimal response rate within 12 treatment cycles based on the International Working Group (IWG) criteria, distinguishing it from the endpoints adopted in trials evaluating JAK inhibitors for approval.Historically, studies on JAK inhibitors predominantly focused on reducing spleen size and alleviating symptom burden as primary endpoints, with each subsequent competitor undergoing evaluation within the same framework.Nonetheless, the prognostic impact of spleen size reduction and symptom alleviation remains ambiguous.Although the COMFORT studies [16,41] demonstrated a survival benefit for Ruxolitinib, despite not being originally powered for this outcome, the definitive disease-modifying potential of these medications remains contentious.Given that allogeneic stem cell transplantation stands as the sole curative option for myelofibrosis, combination therapies should strive for deeper and potentially more clinically significant responses.

Trial status
Protocol Version 4.0 dated 23 Feb 2023.The first subject was enrolled on July 4, 2022.As of May 2024, 23/30 patients have entered the trial and recruitment is planned to be finished with Last-Patient-First-Visit (LPFV) by December 2024.
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Fig. 1
Fig. 1 Study design.Cycle 1 consists of a run-in phase of Fedratinib monotherapy (400 mg QD) followed by addition of Nivolumab (240 mg Q2wk) from cycle 2 on.Patients will receive study treat-

Table 1
Inclusion and Exclusion Criteria Inclusion Criteria Patients must meet ALL of the following inclusion criteria to be eligible for enrollment into the study: a. Signed Informed Consent Form available b. Patients* ≥ 18 years of age c.Patients diagnosed with myelofibrosis (MF) according to the WHO 2008 or 2016 criteria, including primary (pre-fibrotic or overt) and secondary myelofibrosis d.

Table 4
Trials investigating checkpoint-inhibitor treatment in MPN permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.