Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Background: Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Methods: Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. Results: The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. Conclusion: The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

A new dosing regimen of ropeginterferon alfa-2b is highly effective and tolerable: findings from a phase 2 study in Chinese patients with polycythemia vera.

Ropeginterferon alfa-2b represents a new-generation pegylated interferon-based therapy and is administered every 2-4 weeks. It is approved for polycythemia vera (PV) treatment in the United States and Europe with a starting dose of 100 µg (50 µg for patients receiving hydoxyurea) and intra-patient dose titrations up to 500 µg at 50 µg increments, which took approximately 20 or more weeks to reach a plateau dose level. This study aimed to assess ropeginterferon alfa-2b at an alternative dosing regimen with a higher starting dose and quicker intra-patient dose titrations, i.e., the 250-350-500 µg schema, in 49 Chinese patients with PV with resistance or intolerance to hydroxyurea. The primary endpoint of the complete hematologic response rate at treatment weak 24 was 61.2%, which was notably higher than 43.1% at 12 months with the approved dosing schema. The JAK2V617F allele burden decreased from baseline to week 24 (17.8% ± 18.0%), with one patient achieving a complete molecular response. Ropeginterferon alfa-2b was well-tolerated and most adverse events (AEs) were mild or moderate. Common AEs included alanine aminotransferase and aspartate aminotransferase increases mostly at grade 1 or 2 levels. Patients did not present with jaundice or significant bilirubin level increase. No grade 4 or 5 AEs occurred. Seven patients (14.3%) experienced reversible, drug-related grade 3 AEs. No AEs led to treatment discontinuation. Ropeginterferon alfa-2b at the 250-350-500 µg regimen is highly effective and well-tolerated and can help patients achieve greater and rapid complete hematologic and molecular responses.Clinical Trial Registration: This trial is registered at ClinicalTrials.gov (Identifier: NCT05485948) and in China (China National Medical Products Administration Registration Number: CTR20211664).

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Anticancer 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones as tubulin inhibitors.

Studying the anticancer activity of 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones towards cell lines of different cancer types allowed the identification of hit-compounds inhibiting the growth of daunorubicin- (CEM-DNR, IC50 = 0.32-1.28 µM) and paclitaxel-resistant (K562-TAX, IC50 = 0.21-1.23 µM) cell lines, with favorable therapeutic indexes. The studied compounds induced apoptosis and cellular proliferation in treated CCRF-CEM cells. The hit compounds were shown to induce mitotic arrest by interacting with tubulin, inhibiting its polymerization by binding to the colchicine binding site.

SURPASS-ET: phase III study of ropeginterferon alfa-2b versus anagrelide as second-line therapy in essential thrombocythemia.

Patients diagnosed with high-risk essential thrombocythemia (ET) have limited treatment options to reduce the risk of thrombosis and lessen the progression of the disease by targeting the molecular source. Hydroxyurea is the recommended treatment, but many patients experience resistance or intolerance. Anagrelide is an approved second-line option for ET, but concerns of a higher frequency of disease transformation may affect its role as a suitable long-term option. Interferons have been evaluated in myeloproliferative neoplasms for over 30 years, but early formulations had safety and tolerability issues. SURPASS-ET (NCT04285086) is a phase III, open-label, multicenter, global, randomized, active-controlled trial that will evaluate the safety, efficacy, tolerability and pharmacokinetics of ropeginterferon alfa-2b compared with anagrelide as second-line therapy in high-risk ET.

Essential thrombocythemia (ET) is a condition characterized by having more platelets than normal. The high number of platelets increases the risk of a life-threatening blood clot and/or bleeding. Patients with ET and at a high risk for these events are usually treated first with hydroxyurea (HU), but some patients do not respond properly or may develop significant side effects. Anagrelide is an approved medication used in patients who do not respond to HU. Ropeginterferon alfa-2b is a disease-specific, long-acting interferon with a good safety profile approved in polycythemia vera, another type of myeloproliferative neoplasm. The SURPASS-ET clinical trial will evaluate the safety, efficacy, tolerability and pharmacokinetics of ropeginterferon alfa-2b compared with anagrelide in patients with ET who are resistant or cannot tolerate HU. Clinical Trial Registration: NCT04285086 (ClinicalTrials.gov).

Efficacy and safety of ropeginterferon alfa-2b in Japanese patients with polycythemia vera: an open-label, single-arm, phase 2 study.

Ropeginterferon alfa-2b is a novel, site-selective, monopegylated recombinant human interferon alfa-2b. Safety and efficacy of ropeginterferon alfa-2b for the treatment of polycythemia vera were demonstrated in clinical studies conducted in European countries, but clinical studies in Japanese patients are lacking. This phase 2, open-label, multicenter, single-arm study investigated the safety and efficacy of ropeginterferon alfa-2b in 29 Japanese patients with polycythemia vera including young patients and patients with low thrombosis risk who are difficult to receive guideline-based standard treatments. The primary outcome of durable complete hematologic response without phlebotomy at months 9 and 12 was achieved by 8/29 (27.6%) patients. The fastest complete hematologic response was observed at week 12. A corresponding reduction in the JAK2 V617F allele burden from baseline to 52 weeks was also observed (mean ± standard deviation = - 19.2% ± 22.6%). No new safety concerns were identified in Japanese patients when compared with previous studies of ropeginterferon alfa-2b in European populations; the most common treatment-related adverse events were alopecia (55.2%), fatigue (27.6%) and influenza-like illness (27.6%). Most treatment-related adverse events were mild or moderate, with none of grade ≥ 3. Ropeginterferon alfa-2b is a safe and efficacious treatment option in Japanese patients with polycythemia vera.

Hematopoietic expression of a chimeric murine-human CALR oncoprotein allows the assessment of anti-CALR antibody immunotherapies in vivo.

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.

Pharmacokinetics and Pharmacodynamics of Ropeginterferon Alfa-2b in Healthy Japanese and Caucasian Subjects After Single Subcutaneous Administration.

BACKGROUND AND OBJECTIVES: Ropeginterferon alfa-2b is a novel monopegylated recombinant interferon alfa-2b for the treatment of patients with polycythemia vera. The objectives of this study were to evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of ropeginterferon alfa-2b in healthy Japanese subjects compared with Caucasian subjects. METHODS: In this multicenter, parallel-group phase I study, a cohort consisting of six Japanese and six Caucasian subjects was designated to receive a single subcutaneous dose of ropeginterferon alfa-2b (100, 200, 300, and 450 µg). Pharmacokinetic and pharmacodynamic parameters, and immunogenicity were evaluated. Safety was assessed throughout the study. RESULTS: Cohort 4 (450-µg dose) was not initiated because the primary objective of this study was achieved based on the three completed cohorts. A total of 36 enrolled subjects (18 Japanese and 18 Caucasian) in three cohorts were included in the safety, pharmacokinetic, and pharmacodynamic analysis sets. Ropeginterferon alfa-2b exposure in terms of the area under the serum concentration-time curve (AUC) from time zero extrapolated to infinity and the AUC from time zero to the time of the last quantifiable concentration was approximately 1.7-fold and two-fold higher in Japanese subjects than in Caucasian subjects, respectively. Across the same dose range, the maximum serum concentration was approximately 1.25-fold higher in Japanese subjects than in Caucasian subjects. The time to reach the median maximum serum concentration was similar between ethnicities (approximately 96-111 h). The terminal half-life was 48-57 h in Japanese subjects and 31-75 h in Caucasian subjects. The slope of the relationship between dose and drug exposure was greater than 1 in both ethnicities. The dose-dependent induction of beta-2 microglobulin and neopterin expression was observed in both ethnicities, and the two groups showed similar pharmacodynamic parameters. At the end of the study, 22.2% of Japanese subjects and 11.1% of Caucasian subjects developed anti-ropeginterferon alfa-2b-binding antibodies. The neutralizing capacity of these antibodies was not tested. Ropeginterferon alfa-2b up to 300 µg was safe and well tolerated, with no unexpected safety findings based on previous experiences with ropeginterferon alfa-2b and other forms of interferon. CONCLUSIONS: Ropeginterferon alfa-2b exposure was higher in Japanese subjects than in Caucasian subjects. The increase in ropeginterferon alfa-2b exposure was greater than the dose proportion in the dose range of 100-300 µg. Ropeginterferon alfa-2b was safe and well tolerated. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT03546465, registered on 6 June, 2018.

5-Arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones with selective inhibitory activity against some leukemia cell lines.

The data on the pharmacology of 4-thiazolidinones showed that 5-ene-2-(imino)amino-4-thiazolidinones are likely to comprise one of the most promising groups of compounds possessing anticancer properties. A series of 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones was designed, synthesized, and studied against 10 leukemia cell lines, including the HL-60, Jurkat, K-562, Dami, KBM-7, and some Ba/F3 cell lines. The structure-activity relationship analysis shows that almost all tested 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones were characterized by ІС50 values lower or comparable to that of the control drug chlorambucil. Among the tested compounds, (5Z)-5-(2-methoxybenzylidene)- (12), (5Z)-(2-ethoxybenzylidene)- (21), (5Z)-5-(2-benzyloxybenzylidene)- (25), and (5Z)-5-(2-allyloxybenzylidene)-2-(4-hydroxyphenylamino)thiazol-4(5H)-ones (28) possessed the highest antileukemic activity at submicromolar concentrations (ІС50 = 0.10-0.95 µM).

Hematoxylin binds to mutant calreticulin and disrupts its abnormal interaction with thrombopoietin receptor.

Somatic mutations of calreticulin (CALR) have been identified as a main disease driver of myeloproliferative neoplasms, suggesting that development of drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD) abolishes the ability of mutant CALR to oncogenically activate the thrombopoietin receptor (MPL). We therefore hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing a selective growth inhibitory effect on cells that depend on oncogenic CALR for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR-mutated cells than in CALR wild-type cells. Additionally, knockout or C-terminal truncation of CALR eliminated drug hypersensitivity in CALR-mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, disruption of the mutant CALR-MPL interaction, inhibition of the JAK2-STAT5 pathway, and reduction at the intracellular level of mutant CALR upon drug treatment. Our data indicate that small molecules targeting the GBD of CALR can selectively kill CALR-mutated cells by disrupting the CALR-MPL interaction and inhibiting oncogenic signaling.

Ropeginterferon alfa-2b, a novel IFNα-2b, induces high response rates with low toxicity in patients with polycythemia vera.

In this prospective, open-label, multicenter phase 1/2 dose escalation study, we used a next-generation, mono-pegylated interferon (IFN) α-2b isoform, ropeginterferon alfa-2b. The unique feature of ropeginterferon alfa-2b is a longer elimination half-life, which allows administration every 2 weeks. We present data from 51 polycythemia vera patients. The main goal was to define the maximum tolerated dose and to assess safety and efficacy. A dose range of 50 to 540 µg was tested without the appearance of dose-limiting toxicities. All drug-related adverse events were known toxicities associated with IFN-α. The cumulative overall response rate was 90%, comprising complete response in 47% and partial response in 43% of patients; the best individual molecular response level was a complete response in 21% of patients and partial response in 47%. Notably, we did not observe any correlation between the dose level and the response rate or response duration, suggesting that already low levels of ropeginterferon alfa-2b are sufficient to induce significant hematologic and molecular responses. These data suggest promising efficacy and safety of ropeginterferon alfa-2b and support the development of the drug in a randomized phase 3 clinical trial. The study was disclosed at www.clinicaltrials.gov as #NCT01193699 before including the first patient.

Molecular responses and chromosomal aberrations in patients with polycythemia vera treated with peg-proline-interferon alpha-2b.

Fifty-one polycythemia vera (PV) patients were enrolled in the phase I/II clinical study PEGINVERA to receive a new formulation of pegylated interferon alpha (peg-proline-IFNα-2b, AOP2014/P1101). Peg-proline-IFNα-2b treatment led to high response rates on both hematologic and molecular levels. Hematologic and molecular responses were achieved for 46 and 18 patients (90 and 35% of the whole cohort), respectively. Although interferon alpha (IFNα) is known to be an effective antineoplastic therapy for a long time, it is currently not well understood which genetic alterations influence therapeutic outcomes. Apart from somatic changes in specific genes, large chromosomal aberrations could impact responses to IFNα. Therefore, we evaluated the interplay of cytogenetic changes and IFNα responses in the PEGINVERA cohort. We performed high-resolution SNP microarrays to analyze chromosomal aberrations prior and during peg-proline-IFNα-2b therapy. Similar numbers and types of chromosomal aberrations in responding and non-responding patients were observed, suggesting that peg-proline-IFNα-2b responses are achieved independently of chromosomal aberrations. Furthermore, complete cytogenetic remissions were accomplished in three patients, of which two showed more than one chromosomal aberration. These results imply that peg-proline-IFNα-2b therapy is an effective drug for PV patients, possibly including patients with complex cytogenetic changes.