Reference Detail

Ref Type

abstract

PMID

Authors

Matthew C. Stubbs 1 , Hamza Celik 1 , Yanran Ai 1 , Xin He 1 , Hong Chang 1 , Angela Lei 1 , Mark Rupar 1 , Brittney Wass 1 , Melody Diamond 1 , Xiaoming Wen 1 , Jun Li 1 , Nina Zolotarjova 1 , Alex Margulis 1 , Gaurang Trivedi 1 , Angelo B.A. Laranjeira 1 , Cooper A. Taylor 1 , Jennifer Rocha 1 , Christoph Pollmann 2 , Jacob Piehler 2 , Bianca L. Ferreira 3 … Sunkyu Kim 1

Title

Preclinical Evaluation of INCB160058 - a Novel and Potentially Disease-Modifying Therapy for JAK2V617F Mutant Myeloproliferative Neoplasms

Journal	Vol	Issue	Date	Pages	Journal Short Title
Blood	142	Supplement 1	November 2, 2023

URL

https://www.sciencedirect.com/science/article/abs/pii/S0006497123054642

Abstract Text

The Janus kinase 2 ( JAK2) mutation, JAK2V617F, is the most common oncogenic driver in myeloproliferative neoplasms (MPNs), with nearly all cases of polycythemia vera (PV) and over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) patients positive for the somatic mutation. Approved therapies for MPNs such as ruxolitinib, which act by directly inhibiting activity of the kinase domain (JH1) of JAK2, have demonstrated impressive clinical efficacy and safety in patients with MPNs; however, they do not address JAK2V617F allelic burden or achieve molecular remission of disease. A targeted JAK2V617F selective agent sparing wild-type (WT) JAK2 activity has potential to eliminate mutant cells, induce molecular remission, and theoretically lead to functional cure of MPNs. We report herein the preclinical development of pseudokinase (JH2)-targeting INCB160058, a first-in-class, orally bioavailable small molecule with the ability to selectively target JAK2V617F + cell populations derived from patients with JAK-mutant MPNs. Using structure- and function-guided molecular design, INCB160058 was designed to bind with picomolar affinity to the JH2 domain of JAK2V617F at the canonical ATP-binding site, with high specificity (>2500-fold) relative to binding at the active kinase domain (JH1) targeted by currently approved JAK inhibitors. Live cell single-molecule fluorescence microscopy showed that INCB160058 binding to JAK2V617F blocked ligand-independent thrombopoietin receptor dimerization induced by the mutation, and consequently led to loss of JH1 domain kinase activity. X-ray crystallography analysis indicates that the observed inhibition is likely driven by conformational disruption of the ⍺C helix motif at Phe 594 and Phe 595 in conjunction with a shift of the upstream region from Leu 583 to Asn 589 upon INCB160058 binding to the JH2 domain of JAK2V617F. We utilized both CD34 + human multipotent hematopoietic stem cells derived from patients with JAK2-mutant MF, engineered JAK2-mutant human hematopoietic cancer cell lines (eg, SET2 and UKE-1), and murine BA/F3 cell lines to explore the selective effects of INCB160058 on JAK2V617F compared with WT JAK2. INCB160058 treatment selectively reduced pathogenic phospho-STAT5 levels, decreased abnormal megakaryopoiesis, and suppressed colony formation only in JAK2V617F + CD34 + cells but not in CD34 + cells from healthy volunteers. Importantly, continuous exposure of mutant and WT JAK2 cells to INCB160058 in co-cultures at concentrations below IC 50 resulted in progressive elimination of JAK2V617F + cells without affecting WT cells. At the end of the testing period, the JAK2V617F-harboring population was no longer detectable in the co-culture assay. In NSG mice subcutaneously inoculated with JAK2V617F-expressing SET2 cells, INCB160058 was tolerated and exhibited significant antitumor activity. In addition, following INCB160058 treatment, a significant reduction in the engraftment of total human cells, particularly human erythroid progenitors (hCD45 − mCD45 − Ter119 − hCD71 + hCD235a +), was observed in NSGS mice xenotransplanted with JAK2V617F + CD34 + cells. Moreover, INCB160058 treatment also led to the normalization of various pathogenic cytokines, such as interleukin (IL)-6 and IL-8. Importantly, these observations were absent in NSGS mice engrafted with CD34 + cells from healthy volunteers following INCB160058 treatment, further demonstrating the selectivity of INCB160058 for JAK2V617F. In summary, our results indicate a novel mechanism of action of INCB160058, a high-affinity pseudokinase (JH2) binding inhibitor of JAK2V617F that blocks cytokine-independent activity of JAK2V617F while preserving cytokine-dependent signaling. Extended treatment with INCB160058 at low therapeutic doses results in the specific elimination of mutant JAK2V617F-harboring cells in mouse models and human cancer cells with minimal impact on WT counterparts. Clinical testing of INCB160058 may allow patients with MPNs to achieve molecular remission by eliminating cells with the main genetic aberration and afford an opportunity to overcome the disease.

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Molecular Profile	Treatment Approach

Gene Name	Source	Synonyms	Protein Domains	Gene Description	Gene Role

Therapy Name	Drugs	Efficacy Evidence	Clinical Trials
INCB160058	INCB160058	2	1

Drug Name	Trade Name	Synonyms	Drug Classes	Drug Description
INCB160058		INCB-160058\|INCB 160058	JAK2 Inhibitor 19	INCB160058 binds to JAK2 V617F and inhibits dimerization and cytokine-independent kinase activity, which potentially decreases tumor growth (Blood (2023) 142 (Supplement 1): 860).

Gene	Variant	Impact	Protein Effect	Variant Description	Associated with drug Resistance

Molecular Profile	Indication/Tumor Type	Response Type	Therapy Name	Approval Status	Evidence Type	Efficacy Evidence	References
JAK2 V617F	myelofibrosis	predicted - sensitive	INCB160058	Preclinical - Patient cell culture	Actionable	In a preclinical study, INCB160058 decreased Stat5 phosphorylation, abnormal megakaryopoiesis, and colony formation in human hematopoietic stem cells derived from patients with myelofibrosis harboring JAK2 V617F in culture (Blood (2023) 142 (Supplement 1): 860).	detail...
JAK2 V617F	hematologic cancer	predicted - sensitive	INCB160058	Preclinical	Actionable	In a preclinical study, INCB160058 inhibited tumor growth in a mouse model of hematologic cancer expressing JAK2 V617F (Blood (2023) 142 (Supplement 1): 860).	detail...