Inflammation Induced Expression of Ybx1 Mediates Resistance to JAK-Kinase Inhibitor Therapy in Myeloproliferative Neoplasia

Myeloproliferative neoplasia (MPN) is a group of diseases which include polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) and is characterized by clonal proliferation of myeloid cells. In many cases there is also a striking inflammatory syndrome (CRP elevation, fever, elevation of pro-inflammatory cytokines in peripheral blood) in particular in advanced phases of the disease. Auto- and paracrine effects of pro-inflammatory cytokines may contribute to maintenance of the disease and aggravation of the inflammatory syndrome. Cold-shock proteins are multi-functional DNA/RNA-binding proteins and Ybx1 is the prototypic member. These proteins regulate the expression of a number of pro-inflammatory cytokines, as well as cytokine receptors, making them key players in the orchestration of inflammatory processes. Moreover, expression of Ybx1 is induced by cellular and oncogenic stress and mediates resistance to chemotherapy. As patients with MPNs may present with an inflammatory syndrome, we assessed for Ybx1 expression as a potential mediator of cytokine signaling.

By immunohistochemistry we found expression of Ybx1 in primary human bone marrow (BM) biopsies derived from patients with MPN. In contrast, BM sections derived from healthy donors (or remission controls without any evidence for active disease) did not show any expression of Ybx1. Moreover, Ybx1 expression was increased in the cytoplasm of myeloid immune cells derived from the peripheral blood of MPN patients as assessed by flow cytometry. Lower levels of cytoplasmatic Ybx1 were detectable in healthy volunteers.

Therefore, we hypothesized that inflammatory stimuli (e.g. secretion of pro-inflammatory cytokines) may contribute to Ybx1 expression and this may mediate resistance to JAK-kinase inhibitor therapy.

To assess for a functional role of Ybx1 in MPN we used murine cell lines (32D and Ba/F3) transduced with mutated JAK2 (JAK2-V617F) or MPL-receptor (MPLW515L) as well as human cell lines harboring the respective mutations. Exposure of these murine cell lines to pro-inflammatory stimuli (e.g. pro-inflammatory cytokines) induced expression of Ybx1 as assessed by qPCR. As Ybx1-expression has been described to contribute to drug resistance, we aimed to assess for its function in the setting of JAK-kinase inhibitor therapy.

Lentiviral knockdown of Ybx1 alone did neither affect proliferation nor induce apoptosis in JAK2V617F- and MPL-mutated murine cell lines. However, co-treatment with the JAK-kinase inhibitor ruxolitinib (RUX) after Ybx1 knockdown led to induction of apoptosis that significantly exceeded the amount of apoptotic cells (AnnexinV⁺, Caspase⁺) upon RUX-treatment alone. These effects could be re-capitulated in human leukemia cell lines (HEL and SETB2) harboring the JAK2V617F mutation.

As knockdown of Ybx1 (to 40-50% of baseline) alone did not impair proliferation of murine cell lines in vitro, we asked whether reduction of Ybx1 would impair function of normal hematopoiesis. We used a straight knockout mouse model in which homozygous loss of Ybx1 is embryonically lethal. However, heterozygous animals are born without malformations. Ybx1^+/- animals show regular steady state hematopoiesis with Ybx1 expression being reduced to 50%. When injected into lethally irradiated hosts, Ybx1^+/- cells produced a stable chimerism during serial transplantations as compared to Ybx1^+/+ controls.

To assess for a role of Ybx1 in MPN development in vivo, we used a previously published JAK2V617F knock-in mouse model. By the use of a Vav1-Cre-recombinase, a PV-like phenotype was initiated in early hematopoiesis. Sorted LSK-cells from JAK2V617F-knockin-mice were infected with lentiviral Ybx1-shRNA constructs and injected into lethally irradiated hosts. Knockdown of Ybx1 led to a delay in disease onset, when compared to scrambled-shRNA controls. Co-treatment of recipient mice with JAK-kinase inhibitors is currently under way to recapitulate the synergy observed in cell culture models described above.

Taken together, Ybx1 expression in MPN is induced by inflammatory stimuli and protects JAK2V617F or MPLW515L transformed cells from JAK-inhibitor induced apoptosis. Distinct requirement for hematopoiesis (versus disease) offers a therapeutic index to evaluate cold-shock proteins as target structures for inflammatory diseases in the future.