CXCL8/CXCR2 signaling mediates bone marrow fibrosis and represents a therapeutic target in myelofibrosis

Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-kB pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose- dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.

Disseminated tuberculosis with myelofibrosis presentation: a case report

Background Primary myelofibrosis is a rare myeloproliferative disorder in middle-aged and old adults and should be distinguished from secondary and reactive causes of bone marrow fibrosis because, in reactive fibrosis, treatment approaches depend on the underlying etiology. Case presentation Here we report the case of a middle-aged Iranian man who was diagnosed and treated as primary myelofibrosis at presentation, and whose final diagnosis was disseminated tuberculosis with reactive bone marrow fibrosis. Conclusions It is prudent to evaluate the potential causes of myelofibrosis in any patient with the diagnosis primary myelofibrosis. Tuberculosis can be an important etiology of bone marrow fibrosis, especially in endemic areas.

Spleen Stiffness By Magnetic Resonance Elastography or Ultrasound Elastography As a Surrogate Marker of Bone Marrow Fibrosis in Myelofibrosis Patients

Introduction Primary myelofibrosis (PM) is a myeloproliferative neoplasm characterised by bone marrow fibrosis and extramedullary hematopoiesis. Both clinical findings and laboratory parameters are used for prognostic scores in Myelofibrosis patients. In addition, the degree of bone marrow fibrosis has an important prognostic value and has correlation with overall survival. Recently, bone marrow fibrosis was correlated with degree of splenic stiffness (SS) measured by imaging elastography techniques. 1,2 Despite these findings, there were patients with insignificant measures that could not be classified according to marrow fibrosis. In order to advance knowledge in this field, we studied splenic and hepatic stiffness (HS) in patients with myelofibrosis using elastography by two methods, ultrasonography (EUS) and magnetic resonance elastography (MRE), and its correlation with prognostic scores and bone marrow fibrosis. Study Design and Methods This is a prospective, cross-sectional, observational study in patients from the outpatient clinic for myeloproliferative neoplasms who had given informed consent according to procedures approved by institution´s ethical committee. Patients with PM, as well as post-essential thrombocythemia (ET) or post-polycythemia vera (PV) myelofibrosis, were included in this study. Myelofibrosis patients with diagnosis of other associated pathologies that may alter SS, as portal hypertension or cirrhosis, were excluded from the study. Patients were assessed for splenic stiffness measured by ultrasound conducted by two examiners, with more than 10 years of experience. EUS was performed in US Epiq 7 equipment - Philips - with ARFI elastometry methodology. The SS measurements was reported in m/s. In addition, they were also evaluated for splenic and liver stiffness by MRI technique. All exams were performed in 1.5 T MR equipment (Magneton Aera, Siemens Healthineers, Erlangen, Germany) and the MRI protocol included T2-weighted and gradient-echo MRE sequences using steady-state 60-Hz excitation and an external driver placed on the right side and, on the left side of the abdomen. The measures of SS were also obtained by two experienced examiners Results At this moment we present the results of 16 patients with myelofibrosis (PM: 8 cases; post-PV myelofibrosis: 2 cases; post-ET myelofibrosis: 6 cases). The median age was 69y (41-88y) and 62,5% of participants were male. The JAK2 V617F mutation was detected in 9 cases; three cases were CALR positive, and three cases were triple negative. The CBC showed: Hb: 10.9 g/dL (6.5-18.7); WBC (x10 9/L): 9.17 (1.8-44.5) and platelets (x10 9/L): 393 (10-957). Our preliminary results show that bone marrow fibrosis increased according to splenic stiffness by EUS and MRE (Figure 1a; table 1). Patients with osteosclerosis also presented a higher SS by MRE (Figure 1b). We could not find correlation of splenic stiffness with prognostic score DIPSS plus, although Int-2/High risk patients presented a trend to be associated with higher liver stiffness. Conclusion To the moment, our preliminary findings suggest a correlation between SS and degree of bone marrow fibrosis and osteosclerosis, though the correlation between both measures and prognostic scores is still to be determined. We expect to have a better definition for all correlations, as we progress through the assessment of the other patients in our service. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The CXCL1 Inhibitor Reparixin Rescues Myelofibrosis in the Gata1low Model of the Disease

A mayor pathobiological role for interleukin 8 in the etiology of myelofibrosis has been suggested by observations indicating that megakaryocytes expanded in culture from these patients express great levels of interleukin 8 1 and that the plasma levels of this cytokine are predictive of poor prognosis 2. In preliminary experiments we demonstrated that the megakaryocytes from the bone marrow of the Gata1 low model of myelofibrosis express not only high levels of TGF-β, but also levels greater than normal of lipokalin-2, a known inducer of IL-8 production, and of CXCL1, the murine equivalent of IL-8. In addition, these megakaryocytes express also high levels of the CXCL1 receptors CXCLR1 and CXCR2 and the bone marrow from these mice express an CXCR1/CXCR2 activated signature. Using these data as a foundation, we tested here the effects of treatment of Gata1 low mice with the CXCR1/R2 inhibitor reparixin on the myelofibrosis phenotype expressed by this models. To these aim, Gata1 low mice (8-month old) were treated either with vehicle (3 males and 3 females) or with reparixin (formerly referred to as repertaxin) 3 (5 males and 5 females) for either 20 or 37 days. The drug was administered by minipumps implanted subcutaneously in the dorsal region set to deliver 7.5mg of drug/hr/Kg of body weight. The mice receiving the drug for 37 days had the minipumps replaced by day 17. The efficiency of drug delivery decreased over time since the plasma levels of reparixin were 13.90±4.18 and 6.71±4.18ug/mL at day 20 and 37, respectively (p<0.05).The drug was well tolerated with no death or change in body weight recorded over the period of observation. Since the results observed in males and females were similar, the data were pooled for statistical analyses. The treatment did not affect blood values (hematocrit (%): 34.32±3.87 vs 35.63±3.45 and 30.92±3.58, platelets: (x10 3/uL) 187.80±26.12 vs 181.30±53.30 and 99.83±71.92 and white cell counts (x10 3/uL): 2.78±0.55 vs 3.27±0.72 and 3.57±1.43, respectively, in vehicle and day 20- or day 37-reparixin treated mice). The treatment had also little effects on bone marrow (20.55±5.83 vs 22.24±0.85 and 21.68±6.49) and on spleen 141.40±29.04 vs 99.54±15.55 and 173.00±76.54) cellularity. However, the bones were reddish and their sections contained great numbers of erythroid cells, a sign of increased hematopoiesis. Great reductions in the fibrosis of the bone marrow and spleen was observed in mice that had been treated with reparixin compared to vehicle which were statistically significant by day 20 (day 20 bone marrow fibrosis 28.09±15.69 in vehicle and 4.54±0.45 in reparixin treated mice by Gomori, p<0.05; 19.30±7.86 vs 3.19±1.89 by reticulin, staining, p<0.05, respectively by Anova; day 20 spleen fibrosis 20.51±5.25 in vehicle and 10.85±3.82 in reparixin treated mice by Gomori, p<0.05; and 13.15±3.06 vs 6.13±2.34 by reticulin, staining, p<0.05, respectively). Of note when the levels of Gomori and reticulin fibrosis detected at day 20 and 37 in individual mice were inversely correlated with the plasma levels of reparixin observed in the same mice (Figure 1, p<0.01-0.05 by Pearson). Mechanistic insights on these results were provided by Immunostaining of marrow and spleen sections of vehicle and reparixin-treated mice indicating that the megakaryocytes from the reparixin-treated group express levels of TGF-β significantly lower than those expressed by the corresponding cells from vehicle while the levels of LCN-2, CXCL1, CXCR1 and CXCR2 expressed by the reparixin treated megakaryocytes are similar to that of the vehicle treated cells. These results indicate that inhibition of CXCL1 by reparixin, probably by reducing the abnormally high TGF-β content of the megakaryocytes, reduces fibrosis in Gata1 low mice and provide a preclinical rational to test this drug in patients with myelofibrosis. References: 1) Emadi S et al. Blood. 2005;105:464; 2) Tefferi et al, J Clin Oncol. 2011;29:1356; 3) Bertini R et al, PNAS 2004; 101:11791 Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Massucci: Dompe Farmaceutici Spa R&D: Current Employment. Brandolini: Dompe farmaceutici Spa R&D: Current Employment. Giorgio: Dompe farmaceutici Spa R&D: Current Employment. Allegretti: Dompe farmaceutici Spa R&D: Current Employment. Migliaccio: Dompe farmaceutici Spa R&D: Other: received funding for reserach .

IL-1 Signaling Promotes Clonal Expansion and Progression of Bone Marrow Fibrosis in JAK2V617F-Induced Myeloproliferative Neoplasm

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell derived myeloid malignancies characterized by aberrant production of myeloid, erythroid or megakaryocytic lineage cells. JAK2V617F is the most common somatic driver mutation associated with MPN. Interestingly, JAK2V617F mutation can also be detected in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) who do not exhibit overt changes in blood counts. This suggests that other factors might be involved in association with JAK2 mutation in clonal expansion and initiation/progression of MPN. Chronic inflammation is frequently associated with MPN. Interleukin 1 (IL-1) is a major regulator of inflammation. IL-1 consists of two related cytokines IL-1α and IL-1β. Both IL-1α and IL-1β bind to the IL-1 receptor 1 (IL-1R1) to initiate downstream signaling. Although elevated expression of IL-1α and IL-1β has been observed in MPN, their role in the pathogenesis of MPN has remained elusive. In this study, we investigated the role of IL-1 signaling in JAK2V617F-induced MPN using a Jak2V617F knock-in mouse model. We observed elevated levels of IL-1α and IL-1β in mice expressing heterozygous (Jak2 VF/+) and homozygous Jak2V617F (Jak2 VF/VF) compared with WT control animals. Notably, IL-1α and IL-1β expression was significantly higher in Jak2 VF/VF mice exhibiting extensive bone marrow (BM) fibrosis compared with Jak2 VF/+ mice exhibiting polycythemia vera (PV), consistent with elevated levels of IL-1 in patients with myelofibrosis (MF). Since both IL-1α and IL-1β levels were elevated in Jak2 VF/VF mice exhibiting MF, we utilized conditional IL-1R1 knockout (IL-1R1cKO) and Jak2 VF/VF mice to assess the role of IL-1 signaling in the initiation/progression of MF. As expected, Jak2 VF/VF mice exhibited a significant increase in WBC, neutrophil and platelet counts compared to WT control mice. Deletion of IL-1R1in Jak2 VF/VF mice (IL-1R1cKO; Jak2 VF/VF) significantly reduced the WBC, neutrophil and platelet counts to almost control levels. Flow cytometric analysis also showed a significant reduction of myeloid (Gr-1 +) and megakaryocytic (CD41 +) precursors in the BM and spleens of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Moreover, deletion of IL-1R1 significantly reduced hematopoietic stem and progenitor cells (HSPC) in the BM of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Spleen weight was significantly reduced in IL-1R1cKO; Jak2 VF/VF mice compared with Jak2 VF/VF mice and they were comparable to control WT mice. More importantly, deletion of IL-1R1 markedly reduced BM fibrosis in Jak2 VF/VF mice. These data suggest an important role of IL-1 signaling in the progression of BM fibrosis in Jak2V617F-induced MPN. To test whether IL-1 signaling contributes to clonal expansion of JAK2 mutant HSPC, we performed competitive transplantation assays by mixing Mx1Cre; Jak2 VF/+ and Mx1Cre; IL-1R1 F/F; Jak2 VF/+ mice BM cells with CD45.1 + WT mice BM cells at a ratio of 1:1 and transplanted into lethally irradiated CD45.1 + recipient animals. At 4 weeks after BMT, the recipient animals were injected with pI-pC to induce Jak2V617F expression and IL-1R1 deletion. We observed significantly higher percentages of total CD45.2 + cells as well as CD45.2 + myeloid (Gr-1 +), B- and T-cells in the peripheral blood of chimeric mice receiving Jak2 VF/+ BM compared with chimeric mice receiving IL-1R1cKO; Jak2 VF/+ BM. We also observed significantly reduced percentages of CD45.2 + LSK, LK, Gr-1 + and CD41 + cells in the BM of chimeric recipient animals receiving IL-1R1cKO; Jak2 VF/+ BM compared with Jak2 VF/+ BM. These results suggest a role of IL-1 signaling in clonal expansion of Jak2V617F mutant HSPC. Additionally, we tested the effects of blocking IL-1R1 using an anti-IL-1R1 antibody in the homozygous Jak2V617F knock-in mouse model of MF. We observed that anti-IL-1R1 antibody treatment significantly reduced peripheral blood WBC and neutrophil counts and decreased HSPC and myeloid precursors in the BM of Jak2 VF/VF mice. Furthermore, anti-IL-1R1 antibody treatment significantly reduced splenomegaly and markedly reduced BM fibrosis in Jak2 VF/VF mice, suggesting that therapies targeting IL-1R1 could be useful for the treatment of myelofibrosis. Overall, our results suggest that IL-1 signaling contributes to clonal expansion of Jak2V617F mutant HSPC and progression of bone marrow fibrosis in MPN. Disclosures No relevant conflicts of interest to declare.

Dynamic Stromal Changes in Myelofibrosis Patients Pre/Post JAK Inhibition Is Revealed in Clinically Archived Bone Marrow Biopsies By Smooth Muscle Actin (SMA)-CD34 Dual Immunohistochemistry

The natural history of BCR-ABL1 negative myeloproliferative neoplasms (MPNs) is progression towards an overt myelofibrotic (MF) phase with variable risk to develop secondary acute myeloid leukemia. Current treatments include Janus kinase inhibitors (JAKi) which can temporarily alleviate MF-related symptoms but are non-curative and most patients eventually progress to a more advanced stage. Given the negative prognostic impact of bone marrow fibrosis in MPNs and generally poor outcome post JAKi failure, it would be important to identify in situ biomarkers that address the initiation, perpetuation and early reversal of the fibrotic reaction. The current clinical standard for bone marrow fibrosis assessment involves reticulin/trichrome stains that detect relatively static extracellular matrix products rather than the fibrosis driving cells directly. To address this, we have developed a smooth muscle actin stromal-vascular (SMA-CD34) dual immunohistochemical (IHC) technique amenable to morphologic scoring and complemented with a CellProfiler image analysis pipeline. SMA was prioritized over other validated stromal IHC markers given work by others in experimental models demonstrating SMA+ myofibroblasts to be the differentiated output of critical fibrosis inducing Gli1+ 'driver' mesenchymal stem/progenitor cells in MPN. Herein, we demonstrate the feasibility of our translational approach using a clinically annotated cohort of MF patients from the Princess Margaret Cancer Centre MPN Registry. After selecting for high quality (>1.0 cm) paired pre and post JAKi biopsies amenable to image and transcriptome-based analysis, the pilot cohort was comprised of 13 cases with 38% high-risk, 54% intermediate-2 and 8% intermediate-1 risk by DIPSS. Driver mutations were JAK2 V617F (77%), CALR (15%) and other (8%). JAKi therapies included ruxolitinib (31%) + pelabresib (23%), momelotinib (15%), itacitinib (15%) and pacritinib (8%). The SMA-CD34 stromal assessment at baseline revealed distinct interstitial myofibroblast patterns and vascular perturbations not captured by conventional clinical hematopathology assessment (e.g. SMA+ dilated sinusoids). A SMA-CD34 scoring system was developed using a 4-point scale representing normal (0 pts), increased vascularity (1 pt), focal interstitial SMA (2 pts), multifocal interstitial SMA (3 pts) and diffuse SMA (4 pts). Scoring was then performed by blinded hematopathologists. A trend towards JAK2 mutated MF cases demonstrating higher SMA grade at baseline was noted. Interestingly, variable trajectories in SMA scores emerged following treatment with JAKi. Specifically, SMA signals had increased in 15%, decreased in 46% and were stable in 38% post-JAKi when using a morphologic SMA grading scheme. When compared to reticulin fibrosis, the severity of SMA signals had diverged in 1/3 of the cases (e.g. SMA grade decreased, reticulin grade stable). To further complement the SMA-CD34 morphologic grading, a CellProfiler image analysis pipeline was developed yielding a non-vessel associated normalized SMA area metric as a supervised correlate of the clinical SMA scoring system (R 2 = 0.68). Additional supervised and unsupervised bioinformatic approaches for clustering of relevant SMA-CD34 features including an algorithm that informs SMA spatial patterns with respect to niche elements such as arterioles (CD34+SMA+), sinusoids (CD34+) and adipocytes is in development. Lastly, Nanostring Fibrosis V2 panel was employed on a subset that met RNA concentration and quality metrics. Exploratory interpretation showed significant differentially expressed genes in pre vs. post JAKi specimens related to lipid metabolism such as ADIPOR1, SCD, ELOVL6 as well as the chemokine CXCL16. This may suggest a link between fatty acid metabolism and inflammatory differentiation along the SMA-vascular axis in the bone marrow modulated by JAKi treatment. SMA-CD34 IHC stratifies MF bone marrow biopsies differentially from standard WHO reticulin/trichome grading providing a practical formalin-fixed paraffin embedded (FFPE) tissue-based biomarker for assessing fibrosis related bone marrow niche elements from archived clinical samples. While our pilot numbers precluded statistical evaluation by JAKi-type, clinical response and NGS mutational profile at this time, further studies are underway to validate the SMA-CD34 signature on a larger MF cohort. Figure 1 Figure 1. Disclosures Gupta: Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; Incyte: Honoraria, Research Funding; Constellation Pharma: Consultancy, Honoraria; Pfizer: Consultancy.

Final Results of the Fibromet Trial: An Open Label Phase II Study to Evaluate Metformin Effects on Bone Marrow Fibrosis and Disease Progression in Primary Myelofibrosis Patients

Background: Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by myeloid expansion associated with elevation of cytokines involved in fibrosis, angiogenesis, and osteosclerosis, leading to progressive fibrous connective tissue deposition in the bone marrow (BM) and BM failure. Although JAK2, CALR and MPL mutations are frequently seen in PMF, patients' molecular heterogeneity and the lack of an expressive clinical and laboratory response following JAK1/2 inhibitors suggest that other factors, such as unknown protein interactions, additional mutations or epigenetic mechanisms may be involved in the progression of PMF. Metformin (MTF) is an anti-diabetic drug, which has been described to possess anti-cancerous properties through the modulation of the AMPK/TORC1 pathway, thereby causing apoptosis in neoplastic cells. Previous reports demonstrated that MTF significantly reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice. In this context, our goal was to evaluate the effects of MTF treatment in PMF patients. Aims: To report final results of an open label phase II trial (FIBROMET), which evaluated outcomes of PMF patients after 24mo on MTF treatment. Methods: PMF non-diabetic adults were eligible. Patients received MTF in increasing doses until a maximum of 2500mg PO daily, according to tolerance. Primary endpoint was BM fibrosis reversion. Secondary endpoints included reduction of inflammation and downregulation of the JAK-STAT pathway. Samples were collected at the time points: screening (0), 3mo, 6mo, 12mo, 18mo and 24mo. The extent of collagen deposits in BM biopsies was semi-quantitatively assessed with Masson's trichrome stainings, following the recommendations of the European Consensus on grading of BM fibrosis (grades 0, 1, 2 or 3). The levels of CXCL4, sIL-2Ra, IP-10, VEFG-A, MIG, MCP-1, MIP-1b, FGF-2, IL-1RA, IL-5, IL-6, IL-8, IL-15, IL-18, TNFa and TGFb1 were analyzed in BM samples using multiplex assay. Phosphorylation status of intracellular proteins STAT3 and STAT5 was analyzed by flow cytometry and the percentage of cells was recorded using FlowJo software. This trial was approved by the Institutional and National Review Board; written informed consent was obtained from all subjects. REBEC registry number: RBR-52ty66. Results: 11 patients (aged 40-84y) were included between Aug/2018- Feb/2019. Two subjects had early treatment discontinuation due to non-related causes. One patient had disease progression after 12mo of treatment and was submitted to BM transplantation. The median exposure to MTF was 21 mo (3-24) and the median dose was 2500mg/day (1500-2500mg). The most frequent adverse event was diarrhea. No life threatening event occurred. BM collagen deposits were independently evaluated by two hematopathologists with an overall agreement of 80.64% (grade 0: 100%, grade 1: 62.5%, grade 2: 60.0%, grade 3: 90.0%); discordant cases were submitted to joint review. BM collagen deposits did not change when different time points were compared. After 24mo of MTF treatment, a significant 35.4% reduction in IL-5 levels was observed (p=0.03); a 36% reduction in MCP-1 levels was also observed, however this finding was not statistically significant (p=0.06). Flow cytometry analysis demonstrated a STAT3 phosphorylation decrease when comparing screening samples versus 6, 12, 18 and 24 mo of MTF use (all p<0.05), as well as a STAT5 phosphorylation decrease when comparing screening samples versus 6 and 12 mo (all p<0.05). Mean fluorescence intensity for pSTAT3 was: screening 12.01±2.58, 3mo 7.24±1.19, 6mo 4.40±0.24, 12mo 6.31±0.47, 18 mo 6.89±1.01, 24mo 5.71±1.18; and for pSTAT5: screening 15.32±3.59, 3mo 11.50±3.74, 6mo 4.67±0.45, 12mo 6.25±0.58, 18mo 6.24±0.81, 24mo 4.98±0.72. Conclusions: Final results of the FIBROMET trial demonstrated that, in our study population, metformin was not capable of reversing established bone marrow fibrosis in PMF patients. However, a significant downregulation of the JAK-STAT pathway and reduction of cytokine secretion was observed. Finally, metformin showed to be a safe and well-tolerated drug. Phase III studies are required to confirm our results and to evaluate whether MTF treatment in early PMF phases could delay the progression of BM fibrosis through the downregulation of the JAK-STAT pathway. Disclosures Pagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture. OffLabel Disclosure: Metformin for Primary Myelofibrosis treatment.