Abstract: PAC203 is a randomized dose-finding study of pacritinib, an oral JAK2/IRAK1 inhibitor, in patients with advanced myelofibrosis who are intolerant of or resistant to ruxolitinib. Patients were randomized 1:1:1 to pacritinib 100 mg once per day, 100 mg twice per day, or 200 mg twice per day. Enhanced eligibility criteria, monitoring, and dose modifications were implemented to mitigate risk of cardiac and hemorrhagic events. Efficacy was based on ≥35% spleen volume response (SVR) and ≥50% reduction in the 7-component total symptom score (TSS) through week 24. Of 161 patients, 73% were intolerant of and 76% had become resistant to ruxolitinib; 50% met criteria for both. Severe thrombocytopenia (platelet count & lt;50 × 103/μL) was present in 44%. SVR rates were highest with 200 mg twice per day (100 mg once per day, 0%; 100 mg twice per day, 1.8%; 200 mg twice per day, 9.3%), particularly among patients with baseline platelet counts & lt;50 × 103/μL (17%; 4 of 24). Although TSS response rate was similar across doses (100 mg once per day, 7.7%; 100 mg twice per day, 7.3%; 200 mg twice per day, 7.4%), median percent reduction in TSS suggested a dose-response relationship (–3%, −16%, and −27%, respectively). Pharmacokinetic and pharmacodynamic modeling based on all available data showed greatest SVR and TSS reduction at 200 mg twice per day compared with lower doses. Common adverse events were gastrointestinal events, thrombocytopenia, and anemia. There was no excess of grade ≥3 hemorrhagic or cardiac events at 200 mg twice per day. Pacritinib 200 mg twice per day demonstrated clinical activity and an acceptable safety profile and was selected as the recommended dose for a pivotal phase 3 study in patients with myelofibrosis and severe thrombocytopenia. This trial was registered at www.clinicaltrials.gov as #NCT03165734.

Abstract: In patients with cytopenic myelofibrosis, treatment with the JAK2/IRAK1 inhibitor pacritinib was associated with anemia benefit in the phase 3 PERSIST-2 study. The impact of pacritinib on transfusion independence (TI) has not been previously described, nor has the mechanism by which pacritinib improves anemia been elucidated. Because it has been previously postulated that inhibition of activin A receptor, type 1 (ACVR1)/activin receptor-like kinase-2 improves anemia in patients with myelofibrosis via suppression of hepcidin production, we assessed the relative inhibitory potency of pacritinib compared with other JAK2 inhibitors against ACVR1. Pacritinib inhibited ACVR1 with greater potency (half-maximal inhibitory concentration [IC50] = 16.7 nM; Cmax:IC50 = 12.7) than momelotinib (IC50 = 52.5 nM; Cmax:IC50 = 3.2), fedratinib (IC50 = 273 nM; Cmax:IC50 = 1.0), or ruxolitinib (IC50 & gt; 1000; Cmax:IC50  & lt; 0.01). Pacritinib’s inhibitory activity against ACVR1 was corroborated via inhibition of downstream SMAD signaling in conjunction with marked suppression of hepcidin production. Among patients on PERSIST-2 who were not transfusion independent at baseline based on Gale criteria, a significantly greater proportion achieved TI on pacritinib compared with those treated on best available therapy (37% vs 7%, P = .001), and significantly more had a ≥50% reduction in transfusion burden (49% vs 9%, P  & lt; .0001). These data indicate that the anemia benefit of the JAK2/IRAK1 inhibitor pacritinib may be a function of potent ACVR1 inhibition.

Abstract: Introduction: Pacritinib is a JAK2/IRAK1 inhibitor in development for patients with myelofibrosis who have thrombocytopenia. While lower doses of the approved JAK1/2 inhibitor ruxolitinib have been commonly used for the treatment of patients with thrombocytopenia, there is no dosing guidance in the package insert for patients with platelet counts (PLTs) & lt;50 x10 9/L. The starting dose of ruxolitinib in patients with PLTs 50-100 x10 9/L is markedly reduced, which may limit efficacy. Pacritinib, in contrast, was studied without dose attenuation in thrombocytopenic patients (PLTs ≤100 x10 9/L) in the randomized Phase 3 PERSIST-2 study, which showed superior efficacy for pacritinib compared to best available therapy (BAT) based on spleen volume reduction (SVR) and modified total symptom score (TSS) response [Palmer J et al. In submission: ASH abstract 2021]. While many patients in the BAT arm (45%) received ruxolitinib, a comparison between pacritinib and ruxolitinib has not been performed. Here, we conducted a retrospective head-to-head comparison of pacritinib versus ruxolitinib in "first line" (ruxolitinib-naïve) patients treated on PERSIST-2. Methods: PERSIST-2 randomized patients 1:1:1 to pacritinib 200mg BID, pacritinib 400mg QD, or BAT. This analysis focuses on the 200mg BID dose, as 400mg QD is no longer in development, and is restricted to the subgroup of ruxolitinib-naïve patients in PERSIST-2. Patients who received ruxolitinib as BAT prior to week 24 were included; per protocol, ruxolitinib dosing was based on the package insert. Efficacy analyses included the percentage of patients who achieved ≥35% spleen volume reduction (SVR) or ≥50% modified TSS response. Safety analyses were based on all treated patients; efficacy analyses were based on the intention-to-treat population randomized at least 22 weeks prior to study end. The Fisher Exact test was used to describe differences in response rates. Logistic regression was used to adjust for differences in baseline characteristics. Results: Safety analysis included 57 patients on pacritinib 200mg BID and 12 on ruxolitinib; efficacy analysis included 43 on pacritinib and 9 on ruxolitinib. Baseline characteristics were generally similar between the pacritinib and ruxolitinib groups: median age (67 vs. 72), platelet count (51 vs. 49´10 9/L), hemoglobin (9.7 vs. 10g/L), percentage receiving red cell transfusions (37% vs. 33%), and percentage DIPSS high-risk (21% vs. 25%). Differences were noted in the percentage of patients with ≥1% peripheral blasts (30% vs. 75%) and with primary MF (75% vs. 58%). Most patients treated with pacritinib were able to maintain full doses over time. By contrast, patients on ruxolitinib received a median starting dose of 10mg daily (inter-quartile range [IQR]: 10, 10mg] at baseline, 10mg daily at week 12 [IQR: 0, 10mg], and 10mg daily at week 24 [IQR: 0, 20] (Figure 1). Patients treated with pacritinib had numerically higher rates of SVR (28% vs. 11%) and modified TSS response (37% vs. 11%). The hazard ratio for overall survival for pacritinib vs. ruxolitinib was 0.49 [95% CI: 0.13-1.92]. There was no diminution of treatment effect observed for SVR, TSS, or survival after adjusting for baseline age, DIPSS risk, platelet count, and primary vs. secondary MF. Nearly all patients on pacritinib and ruxolitinib experienced AEs (93% vs. 100%). The most frequent AE was diarrhea, mainly grade 1-2, which occurred more on pacritinib versus ruxolitinib (47% vs. 8%). Cytopenias with full-dose pacritinib occurred at similar rates as with low-dose ruxolitinib (thrombocytopenia: 33% vs. 33%; anemia: 30% vs. 25%). Hemorrhagic events occurred at similar rates on pacritinib and ruxolitinib (all grade: 44% vs. 58%; grade ≥3: 19% vs. 17%). Cardiac events occurred at similar rates on pacritinib and ruxolitinib (26% vs. 33%; grade ≥3: 5% vs. 17%). Infection of any grade occurred more frequently on pacritinib (47% vs. 33%), while grade ≥3 infections were less common (11% vs. 17%). Fatal AEs were more common with ruxolitinib (7% vs. 25%). Conclusion: Full-dose pacritinib yielded higher response rates and a similar safety profile to lower doses of ruxolitinib in "first-line" patients with MF who have moderate or severe thrombocytopenia. Figure 1 Figure 1. Disclosures Mascarenhas: Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Promedior: Consultancy, Membership on an entity's Board of Directors or advisory committees; Prelude: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Merus: Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Galecto: Consultancy; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Forbius: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bose: BMS: Honoraria, Research Funding; NS Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; CTI BioPharma: Honoraria, Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Honoraria, Research Funding; Sierra Oncology: Honoraria; Kartos Therapeutics: Honoraria, Research Funding; Novartis: Honoraria; Blueprint Medicines: Honoraria, Research Funding; Promedior: Research Funding. Kiladjian: Taiho Oncology, Inc.: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Mesa: CTI: Research Funding; Abbvie: Research Funding; Novartis: Consultancy; Incyte Corporation: Consultancy, Research Funding; Pharma: Consultancy; Constellation Pharmaceuticals: Consultancy, Research Funding; Genentech: Research Funding; Sierra Oncology: Consultancy, Research Funding; AOP: Consultancy; Gilead: Research Funding; Promedior: Research Funding; Samus: Research Funding; CTI: Research Funding; La Jolla Pharma: Consultancy; Celgene: Research Funding. Gerds: Novartis: Consultancy; PharmaEssentia Corporation: Consultancy; Constellation: Consultancy; CTI BioPharma: Research Funding; Sierra Oncology: Consultancy; AbbVie: Consultancy; Celgene/Bristol Myers Squibb: Consultancy. Gupta: BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Constellation Pharma: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy. Harrison: 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; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Speakers Bureau; Geron: 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; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: 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; AOP Orphan Pharmaceuticals: 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; 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. Yacoub: Agios: Membership on an entity's Board of Directors or advisory committees; Acceleron Pharma: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau. Garcia Gutierrez: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Vannucchi: Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Scheid: Roche: Consultancy; Abbvie: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Buckley: CTI Biopharm: Current Employment. Roman-Torres: CTI Biopharm: Current Employment. 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. Sobas: Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Verstovsek: CTI BioPharma: Research Funding; Celgene: Consultancy, Research Funding; Genentech: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Protagonist Therapeutics: Research Funding; Ital Pharma: Research Funding; Incyte Corporation: Consultancy, Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; PharmaEssentia: Research Funding; Roche: Research Funding; AstraZeneca: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy.

Abstract: Mutations in CSF3R (aka GCSFR) occur in the majority of patients with Chronic Neutrophilic Leukemia (CNL) and also are rarely found in adult and pediatric Acute Myeloid Leukemia. The most common CSF3R mutation in CNL is T618I (aka T595I), a point mutation in the membrane-proximal extracellular domain that causes ligand independence. Mutations that lead to a premature stop in the cytoplasmic domain are also found in CNL and result in increased expression of CSF3R on the cell surface. Understanding the biology of other novel mutations in CSF3R may lead to insight into both receptor biology and oncogenesis. The CSF3R N610H mutation was identified in a patient with a myeloproliferative neoplasm, which was most consistent with a JAK2, CALR, MPL mutation negative primary myelofibrosis. This patient had a history of mild leukocytosis for several years with most recent white blood cell counts between 13.3 and 15.3 x 103/uL. A bone marrow biopsy revealed 90% cellularity with mildly increased reticulin fibrosis, increased myeloid to erythroid ratio, no overt dysplasia, and less than 5% blasts. The cells were karotypically normal with a micro deletion of the 3' end of PDGFRB (5q) identified by FISH at 59%. The patient has had minimal symptoms with no anemia or thrombocytopenia and is currently being monitored but not receiving any intervention. The patient's bone marrow was next-generation sequenced using a 42-gene myeloid malignancy targeted mutation hotspot panel which revealed a mutation at N610H in CSF3R at a 50% mutant allele frequency. Given its proximity to the most common CSF3R mutation found in Chronic Neutrophilic Leukemia (T618I, aka T595I), we were interested in understanding whether the N610H mutation might contribute to disease biology. N610 (also known as N586 in the traditional numbering system that does not include the signal peptide) is part of an N-X-T motif, which is a consensus sequence for N-linked glycosylation. Haniu et al (Biochemistry 1996) demonstrated that N610 is one of 8 sites that are N-glycosylated on CSF3R. We confirm by mass spectrometry (MS) based analysis that N610 is occupied with a bisecting complex N-glycan (in vitro). We further found that the N610H mutation and a more conservative N610Q substitution are highly activating in CSF3R, leading to cytokine-independent growth in the murine Ba/F3 cell line. Furthermore, like the T618I mutation, these mutations render the receptor ligand-independent. N610H and N610Q lead to a robust increase in downstream signaling through the JAK/STAT pathway as demonstrated by an increase in the levels of phospho-STAT3. The loss of N-glycosylation in the membrane-proximal region of CSF3R may therefore increase ligand-independent receptor activation and promote oncogenesis. This study highlights the insight that rare human mutations can provide into the relationship between receptor structure and function. Disclosures Radich: Ariad: Consultancy; Novartis: Consultancy, Research Funding; Gilliad: Consultancy; Incyte: Consultancy. Bertozzi:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Catalent Biologics: Membership on an entity's Board of Directors or advisory committees; Verily: Membership on an entity's Board of Directors or advisory committees; Enable Bioscience: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Oh:CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: PAC203 is a global multicenter dose-finding study of pacritinib (PAC), an oral JAK2/IRAK1 inhibitor in patients with primary or secondary myelofibrosis refractory or intolerant to ruxolitinib, including patients with severe thrombocytopenia. Patients were randomized 1:1:1 (PAC 100mg QD, 100mg BID or 200mg BID) and stratified by baseline platelet count. The mutational landscape of this patient group is not well characterised, and the impact of mutation status on disease response and hematologic parameters is unknown. We carried out baseline mutational analysis on 105 (of total 164 recruited; 161 treated) patients using an ISO accredited Illumina TruSeq Custom Amplicon Panel, including 32 gene mutation hotspots and exons (~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. Median follow-up time for this cohort was 163 (28-476) days. 57% of patients had a diagnosis of primary myelofibrosis (PMF), whereas 27% and 15% had post-polycythemia vera MF (PPV-MF) and post-essential thrombocythemia MF (PET-MF), respectively. The median baseline platelet count was 64.5 x109/L, with 〈 50 x109/L in 38.3% of patients and baseline Hb 〈 10g/dL in 67.8% of patients. The median age was 67.5 (37-87) years. The majority of patients were JAK2 V617F-mutated (77%), followed by MPL-mutated (8.6%), with a notably low incidence of CALR-mutated (8.6%; type 1 CALR n=6, type 2 CALR n=3) and "triple-negative" (4.8%) cases. Non-myeloproliferative neoplasm driver mutations (NDM) were present in 77.1% (n=81) with ≥3 NDMs in 18.1% of patients. Similar to previous reports, the most prevalent NDMs were TET2 and ASXL1 (n=27 and n=25 respectively). Splicing factor (SF) mutations were mutually exclusive and detected in 32.3% of patients: SF3B1 [13], U2AF1 [14] , SRSF2 [6], ZRSR2 [1] . RAS pathway mutations (KRAS/NRAS) were found at a higher frequency than previously in MF cohorts; 16.2% (n=17). SF mutations were present in more PMF (45.6%) than PPV-MF (3.7%, n=1/27) or PET-MF patients (33.3%, n=5/15), P=.002. SF3B1-mutated patients had higher trial entry platelet counts (66.7% platelets ≥100x109/L, P=.014). Baseline red cell transfusion dependency was more often associated with U2AF1 mutations 24.1% (n=7/29) as compared with red cell transfusion independence, 4.5% (n=2/44), P=.047 and U2AF1-mutated patients had lower baseline hemoglobin 〈 10g/dL (n=11/12), P=.015. Overall, 41 (39%) of patients had a high molecular risk mutation (HMR; IDH1/2, SRSF2, ASXL1, SRSF2, U2AF1Q157), and 8 patients had TP53 mutations. In those with molecular analysis available, the highest rates of ≥35% SVR were observed in the 200mg BID arm: 11.1% (n=4/36) followed by 3.2% (n=1/32) in 100mg BID arm and 0% (n=0/30) in 100mg QD arm). Rates of TSS reduction ≥50% were similar across arms. There were no significant correlations between mutations and SVR or TSS response. More grade 3/4 anemia occurred in TET2-mutated patients (OR 5.7, 95% CI 1.6-20.4, P=.007). RAS pathway mutations were associated with grade 3/4 thrombocytopenia (OR 4.4, 95% CI 1.3-14.8, P=0.016). Treatment discontinuation was not influenced by mutation status. In summary, the PAC203 cohort is molecularly high risk, with a high incidence of HMR and low incidence of CALR mutations. We identified novel associations between mutation profiles and hematologic events in this population with advanced MF. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership. Gerds:Roche: Research Funding; Incyte: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding; Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Celgene Corporation: Consultancy, Research Funding. Buckley:CTI BioPharma: Employment, Equity Ownership. Harrison:Incyte: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Promedior: Honoraria; Celgene: Honoraria, Speakers Bureau; AOP: Honoraria; Sierra Oncology: Honoraria; Janssen: Speakers Bureau; Gilead: Speakers Bureau; Roche: Honoraria; CTI: Speakers Bureau; Shire: Speakers Bureau. Oh:Novartis: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees. Mead:CTI: Honoraria, Research Funding; Bristol Myers-Squibb: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Pfizer: Consultancy.

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.

Abstract: Introduction: Thrombocytopenia, a hallmark of cytopenic myelofibrosis (MF), is associated with poor survival and quality of life impairment. Patients with MF and moderate or severe thrombocytopenia (platelet counts & lt;100x10 9/L, & lt;50x10 9/L) tend to have high symptom burden as measured by the Total Symptom Score (TSS), driven largely by physical functioning symptoms (Mesa R et al, ASH 2021 abstract in submission). Pacritinib, an investigational JAK2/IRAK1 inhibitor, was studied in patients with platelet counts ≤100x10 9/L in the PERSIST-2 trial. Unlike the pivotal studies upon which available JAK1/2 inhibitors were approved that relied on a modified TSS version that excluded 'tiredness' from the response analysis, PERSIST-2 included 'tiredness' as part of TSS and found response rates of 25% for pacritinib vs. 14% for best available therapy (BAT), P=0.08. Here, we retrospectively analyzed TSS on PERSIST-2 using the modified scoring system. In addition, we evaluated the impact of pacritinib and BAT, including ruxolitinib, on MF symptoms including 'tiredness' and 'inactivity'. Methods: All PERSIST-2 patients in the intention-to-treat population randomized at least 22 weeks prior to the end of the study were included. PERSIST-2 randomized patients 1:1:1 to pacritinib 200mg BID, pacritinib 400mg daily, or BAT (including ruxolitinib). The modified TSS score was calculated as the sum of individual symptom scores for early satiety, abdominal discomfort, rib pain, night sweats, itching, and bone pain ('tiredness' and 'inactivity' were not included), and response was defined as a ≥50% reduction in score from baseline to week 24. Symptoms were categorized as physical function-related (tiredness, inactivity), spleen-related (satiety, abdominal discomfort, rib pain), or cytokine-related (sweats, itching, bone pain). The Wilcoxon Rank Sum test was used to compare differences in scores. Results: The analysis included 74 patients randomized to pacritinib 200mg BID and 72 to BAT; an additional 75 were randomized to pacritinib 400mg QD (dose no longer in development). At baseline, 39% had moderate and 46% had severe thrombocytopenia. Ruxolitinib was selected as BAT in 45% of patients with moderate and 38% with severe thrombocytopenia. The co-primary endpoint for the study - TSS response rate in pooled pacritinib arms versus BAT - showed a significant difference using the modified TSS: 30% vs. 14%, P=0.008. Similarly, the modified TSS response rate was higher for patients treated with pacritinib 200mg BID compared to BAT (35% vs. 14%, P=0.004, Figure 1a). Among patients on the BAT arm, modified TSS response rates were modestly higher for patients who received ruxolitinib prior to week 24 (19% [n=6/32]) compared to those who did not (10% [n=4/40] ), though both were lower than response rates with pacritinib. Patients on the pacritinib 200mg BID arm experienced greater percentage reductions in individual myelofibrosis symptoms between baseline and week 24 compared to BAT, both in patients with moderate and severe thrombocytopenia (Figure 1b, 1c). For physical function symptoms, severity was reduced more on pacritinib 200mg BID compared to BAT by week 24 (median reduction in tiredness: 30% vs. 13%, P=0.0261; inactivity: 22% vs. 7%, P=0.099). Among patients with severe thrombocytopenia, symptom improvement was minimal on BAT (Figure 1b). Conclusion: The symptom benefit of pacritinib in patients with moderate and severe thrombocytopenia is demonstrated in the PERSIST-2 study. A significant reduction in symptoms was observed in patients treated with pacritinib 200mg BID, the dose being studied in an ongoing Phase 3 trial (PACIFICA), compared to BAT. Furthermore, the modified TSS response rate of 35% in patients treated with pacritinib 200mg BID is comparable to that reported for approved JAK1/2 inhibitors in patients with higher platelet counts, suggesting the potential for pacritinib to address the unmet need of patients with cytopenic myelofibrosis. Figure 1 Figure 1. Disclosures Palmer: CTI BioPharma: Consultancy, Research Funding; Protagonist: Consultancy, Research Funding; Incyte: Research Funding; PharmaEssentia: Research Funding; Sierra Oncology: Consultancy, Research Funding. Mesa: Incyte Corporation: Consultancy, Research Funding; Samus: Research Funding; Sierra Oncology: Consultancy, Research Funding; CTI: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Pharma: Consultancy; La Jolla Pharma: Consultancy; Constellation Pharmaceuticals: Consultancy, Research Funding; CTI: Research Funding; Abbvie: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Promedior: Research Funding; AOP: Consultancy. 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. Rampal: Sierra Oncology: Consultancy; Novartis: Consultancy; BMS/Celgene: Consultancy; Stemline: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Constellation: Research Funding; Kartos: Consultancy; Disc Medicine: Consultancy; Blueprint: Consultancy; Pharmaessentia: Consultancy; Incyte: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment; Abbvie: Consultancy; CTI: Consultancy. Buckley: CTI Biopharm: Current Employment. Roman-Torres: CTI Biopharm: Current Employment. Verstovsek: Protagonist Therapeutics: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Incyte Corporation: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; NS Pharma: Research Funding; AstraZeneca: Research Funding; Blueprint Medicines Corp: Research Funding; CTI BioPharma: Research Funding; Promedior: Research Funding; Ital Pharma: Research Funding; PharmaEssentia: Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. Mascarenhas: Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merus: Research Funding; Geron: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Forbius: Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy, Research Funding; Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Consultancy; Galecto: Consultancy; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vannucchi: AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kiladjian: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; AP Orphan: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees. Harrison: Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Speakers Bureau; Roche: 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; BMS: 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; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: 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; Sierra Oncology: Honoraria; Gilead Sciences: 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; CTI BioPharma: 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. Bose: Novartis: Honoraria; NS Pharma: Research Funding; Pfizer: Research Funding; Constellation Pharmaceuticals: Research Funding; Blueprint Medicines: Honoraria, Research Funding; Kartos Therapeutics: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Promedior: Research Funding; CTI BioPharma: Honoraria, Research Funding; Sierra Oncology: Honoraria; Astellas: Research Funding; BMS: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Craig: CTI BioPharma: Current Employment. Gerds: PharmaEssentia Corporation: Consultancy; CTI BioPharma: Research Funding; Sierra Oncology: Consultancy; AbbVie: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Constellation: Consultancy; Novartis: Consultancy.