Evidence of NF-ΚB Pathway Activation in Patients with Advanced, High Molecular Risk Myelofibrosis
Authors: O'Sullivan Jennifer , Gerds Aaron T. , Harrison Claire N. , Oh Stephen T. , Hamblin Angela , Buckley Sarah A , List Alan F. , Mead Adam J. 
Published: November 2021

Abstract
Introduction: Patients with myelofibrosis who discontinue treatment with the JAK1/2 inhibitor ruxolitinib have a poor prognosis that is often associated with advanced phases of disease and severe cytopenias. While these patients are more likely to have high molecular risk genomic markers, biological drivers of disease in this advanced population are not well characterized. PAC203, a Phase 2 dose-finding study in patients with symptomatic myelofibrosis who were intolerant of or resistant to ruxolitinib, represents an opportune cohort for analyzing the association between high molecular risk (HMR) mutations and disease phenotype. Patients had advanced disease at study entry, with profound cytopenias and high mutational burden [O'Sullivan J et al. Blood (2019) 134 (Sup_1):4214.]. Here, we analyzed the interaction between high-risk mutations and cytokine profiles of patients treated in PAC203.
Methods: Cytokine and mutation data were available in 108 (of total 164 recruited; 161 treated) patients. Using the Myriad RBM platform, a microsphere-based immuno-multiplexing technology, 47 cytokines were assessed. Mutation profiles were determined using an ISO accredited Illumina TruSeq Custom Amplicon Panel, composed of mutational-hotspots/exons from 32 genes (~36,000 bp, 287 amplicons). CALR mutation screening was carried out independently. Accepted coverage was achievement of a depth of ≥100 reads per base in ≥95% of targeted bases. The initial analysis assessed possible relationships between individual plasma cytokine levels and specific somatic gene mutation and clinical demographic data. An unsupervised approach was then used applying hierarchical agglomerative clustering to identify related sets of cytokines. Associations between cluster scores, based on the median overall cytokine concentration within each cluster for each patient, and clinical and genomic data was assessed.
Results: The median baseline platelet count was 64 x10 9/L (38% with platelets &lt;50 x10 9/L) and baseline Hb &lt;10g/dL in 68% of patients. The median age was 68 (37-87) years. The mutation profile of this cohort was previously described, with JAK2 V617F mutations (78%) the most prevalent driver mutation, followed by CALR mutations (13%), MPL mutations (7%), and patients were "triple-negative" in 2% of cases. Non-MPN driver mutations (NDM) were present in 76%, most commonly mutated-ASXL1 and -TET2 (27% and 24% respectively). Overall, 41% of patients had a high molecular risk mutation (HMR; IDH1/2, SRSF2, ASXL1, SRSF2, U2AF1Q157), splicing factor (SF) gene mutations were detected in 32% of patients and RAS pathway mutations (KRAS/NRAS) were found at a higher frequency than previously in MF cohorts (18%).
Analysis of cytokine data using unsupervised learning identified 6 clusters (Figure 1). Among these, elevations in clusters 2 (p=0.009) and 4 (p=0.006) were associated with presence of HMR mutations. Higher cluster 2 scores were also associated with driver mutation variant allele frequencies≥50%, p=0.007. Notably, the pro-inflammatory cytokines in cluster 2 linked to HMR mutations (HMR+) represented a transcriptional cluster regulated by the NF-κB pathway. The presence of a HMR mutation was associated with higher IL-8 levels (40.5pg/ml) as compared with absence (24.5pg/ml), p&lt;0.0001. Elevated tumour necrosis factor-alpha (TNF- α) and IL-18 levels were also associated with HMR mutations; TNF-α 61pg/ml in HMR+ vs. 48.5pg/ml for HMR-. Although RAS-pathway mutations were not associated with a specifc cluster scores, these patients did have higher levels of the NF-kB-associated cytokine IL12P40 (1.1ng/ml) as compared with RAS-pathway wild-type patients (0.6ng/ml), p=0.001. There was no association between cytokine cluster scores and recent exposure to RUX at trial entry.
Conclusions: In this high-risk cohort of previously RUX-treated MF patients enriched for HMR and RAS-pathway mutations, we report for the first time a relationship between HMR somatic gene mutations and an NF-κB directed pro-inflammatory cytokine signature, implicating the activation of a distinct biological signaling pathway operative in this molecularly-defined cohort.
Figure 1 Figure 1.

Disclosures
Gerds: CTI BioPharma: Research Funding; AbbVie: Consultancy; Sierra Oncology: Consultancy; PharmaEssentia Corporation: Consultancy; Constellation: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Harrison: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Oh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees. Buckley: CTI Biopharm: Current Employment. List: CTI Biosciences: Consultancy; Halia Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company; Precision BioSciences: Current Employment, Current equity holder in publicly-traded company; Aileron Therapeutics: Consultancy. Mead: Celgene/BMS: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Speakers Bureau.