The Deubiquitylase Usp22 Is Important for KRAS-Driven Myeloproliferative Disease

Mutations in Ras pathway are among the most common gene abnormalities in several types of cancer including hematological malignances. Ras gene (NRAS or KRAS) mutations are found in 20-40% of myeloid malignances and in 15-30% of acute lymphoblastic leukemia. Mutations in KRAS are the second most frequently mutated. Expression of the most common KRAS mutation (G12D) in mouse hematopoietic cells causes a fatal myeloproliferative neoplasm (MPN). The disease observed in these mice resembles the human MPNs juvenileand chronic myelomonocytic leukemias characterized by leukocytosis, splenomegaly and anemia. Patients with these MPNs typically survive for 10 months to 4 years and are still considered incurable.

Previous studies comparing mouse and human samples led to the identification of USP22 (Ubiquitin Specific Peptidase 22) as part of a self-renewal gene expression signature in different types of cancer including hematological malignances. We analyzed the publicly available database, Oncomine, and showed that USP22 is highly expressed in acute myeloid and lymphoid leukemias. Moreover, USP22 was found as part of an oncogenic network in acute myeloid leukemia with mutations in an upstream activator of Ras signaling. Thus, to study a possible role of Usp22 in Ras-induced myeloproliferative disease, we generated Kras^G12D/+ Mx1^Cre/+ (hereby named KM), Kras^G12D/+ Mx1^Cre/+ Usp22^F/F (KMU) and Mx1^Cre/+ Usp22^F/F (MU) mice.

Analysis of peripheral blood cells showed no changes in blood cell counts in MU mice compared to those of control mice, indicating that Usp22 deletion does not affect steady state hematopoiesis. As expected, KM mice had an increase in neutrophils and monocytes as previously reported. In contrast, KMU mice showed a dramatic decrease in neutrophil counts, suggesting that USP22 is possibly involved in KRAS-induced myeloproliferative disease.

Analysis of bone marrow populations showed no significant changes in the frequency of HSCs, GMP, CMP or MEP populations between KM and KMU mice. However, there was a statistically significant decrease in the Gr1⁺ CD11b⁺ population in KMU compared to KM mice. This decrease was mostly associated with a decrease in Gr1 expression and accumulation of CD11b⁺ cells. We also performed studies using colony-forming assays in methylcellulose media. Bone marrow cells from KM mice were able to form colonies in the absence of cytokines as previously reported. However, Usp22 deletion (KMU mice) decreased cytokine-independent growth by more than 50%.

Previous reports have shown that transplantation of KM bone marrow cells lead to the generation of myeloproliferative disease and thymic lymphomas in recipient mice. To investigate whether Usp22 deletion could limit the generation of these malignances, we transplanted bone marrow cells from KM and KMU mice into lethally irradiated mice. Although KMU recipient mice died earlier than KM recipient mice for unknown reasons, myeloproliferative disease was observed in only 25% of KMU recipient mice compared to 75% of KM recipient mice. Moreover, the thymus size of KMU recipient mice was statistically significant smaller than those of KM recipient mice.

In summary, we found that Usp22 may play an important role in Kras-driven myeloid and lymphoid malignances. Usp22 deletion in KM mice induced a dramatic decrease in neutrophil counts, decreased numbers of Gr1⁺ CD11b⁺ cells in the bone marrow and decreased cytokine-independent growth of KM bone marrow cells. Moreover, transplantation of KMU cells into lethally irradiated recipients resulted in decreased generation of myeloid and lymphoid malignances. We are currently performing additional studies to dissect the molecular mechanism by which Usp22 affects Kras-driven hematological malignances.