Abstract
Pegylated Interferon alpha (PEG-IFNa) has proven clinical and molecular efficacy in the treatment of Bcr-Abl negative myeloproliferative neoplasms. Indeed PEG-IFNa induces complete hematological remissions but also molecular responses demonstrated by the reduction of the JAK2V617F (JAK2VF) allele burden. AOP2014/P1101 is a next generation long-acting PEG-IFNa-2b, recently shown to be safe and well tolerated in phase I-II studies in PV patients (pts) (Gisslinger at al, 2012). In addition, molecular responses (MR) were reported in these early phase trials with 50% of pts achieving partial MR at month 18. Furthermore, one pt with a baseline JAK2VF allelic burden of 22% achieved complete MR after 36 weeks of therapy suggesting a specific effect of AOP2014/P1101 on JAK2VF-positive cells.
To study the differential impact of AOP2014/P1101 on the proliferation and differentiation of wild type versus JAK2VF hematopoietic cells.
We studied the effects of AOP2014/P1101 at different concentrations consistent with assumed exposure in patients (0.5 and 2µg/ml), compared with standard recombinant interferon alpha-2a (rIFNa-2a) at 700 U/ml in human cell lines in liquid suspension assay, and in normal and PV samples in methylcellulose assays. A) Two different human JAK2VF cell lines were studied: HEL characterized by the presence of multiple copies of JAK2VF, and UKE-1 which harbors 2 copies of JAK2VF. Cell lines were grown in presence or absence of drugs and living cells were counted each day during 3 days. B) Normal hematopoietic progenitors derived from cord blood and primary cells from 4 PV patients were studied in clonogenic assays (Methocult, Stemcell technologies©). In PV pts samples the presence of endogenous erythroid colonies (EECs) was determined in cultures without erythropoietin (EPO). Genotyping of the colonies was performed by picking the colonies, extracting the DNA and testing for the presence of JAK2VF using the JAK2 Mutascreen kit (Qiagen©).
In both HEL and UKE-1 JAK2 mutated cell lines AOP2014/P1101 exhibited a dose-dependent anti-proliferative effect which was comparable to that of rIFNa-2a. AOP2014/P1101 at 0.5µg/ml and 2µg/ml induced a 9%, and 41% inhibition of HEL cells proliferation, respectively (resp). Same doses induced an 18% and 35% inhibition of UKE-1 cells proliferation, resp. A similar antiproliferative effect was observed with rIFNa-2a with 38%, and 36% inhibition of HEL, and UKE-1 cells proliferation, resp.
In normal CD34+ hematopoietic progenitors derived from cord blood, AOP2014/P1101 induced a small decrease in the number of erythroid colonies (median reduction by 7%, 15% and 14% with AOP2014/P1101 0.5 µg/ml, 2 µg/ml, and rIFNa-2a, resp). Myeloid colonies were almost unaffected by AOP2014/P1101 (no modification at 0.5µg/ml, and 9% reduction at 2µg/ml) while a reduction by 18% was obtained using rIFNa-2a, confirming the low level of toxicity of AOP2014/P1101 on normal hematopoietic progenitors.
In contrast, AOP2014/P1101 drastically reduced the growth of JAK2VF erythroïd progenitors in all the PV samples tested. A 41%, and 62% median reduction of the number of EPO-stimulated colonies was observed with AOP2014/P1101 0.5µg/ml, and 2µg/ml, respectively. A more striking effect was observed on colonies grown in the absence of EPO: the number of EECs was reduced by 82% with AOP2014/P1101 2µg/ml compared to untreated cells. This result suggested that AOP2014/P1101 inhibited more efficiently JAK2-mutant hematopoietic progenitors. This hypothesis was confirmed by the study of the JAK2 genotype at the clonogenic level: a median 2-fold increase in the ratio of wild type to mutant colonies with 2µg/ml AOP2014/P1101 was observed, suggesting that AOP2014/P1101 does specifically target PV erythroid progenitors harboring the JAK2VF mutation. In addition, in one patient with both homozygous and heterozygous JAK2VF colonies, eradication of homozygous colonies was achieved with AOP2014/P1101, suggesting that JAK2VF homozygous progenitors are even more sensitive to AOP2014/P1101.
Our results show that AOP2014/P1101, a novel form of PEG-IFNa-2b, can specifically target PV JAK2VF hematopoietic progenitors while sparing wild type cells. These results are in line with the clinical efficacy with low hematologic toxicity reported in early phase trials with AOP2014/P1101.
Cassinat:AOP Orphan: Research Funding. Klade:AOP Orphan Pharmaceuticals AG: Employment. Zahriychuk:AOP Orphan Pharmaceuticals AG: Employment. Kiladjian:AOP Orphan: Honoraria, Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
