Phosphorylation of BECLIN-1 by BCR-ABL suppresses autophagy in chronic myeloid leukemia.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL+ cells.

Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms.

Background: Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance. Methods: Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software. Results: In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors. Conclusion: Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.

Oncogenic JAK2V617F requires an intact SH2-like domain for constitutive activation and induction of a myeloproliferative disease in mice.

The oncogenic JAK2V617F mutation is found in myeloproliferative neoplasms (MPNs) and is believed to be critical for leukemogenesis. Here we show that JAK2V617F requires an intact SH2 domain for constitutive activation of downstream signaling pathways. In addition, there is a strict requirement of cytokine receptor expression for the activation of this oncogene. Further analysis showed that the SH2 domain mutation did not interfere with JAK2 membrane distribution. However, coimmunoprecipitated experiments revealed a role for the SH2 domain in the aggregation and cross-phosphorylation of JAK2V617F at the cell membrane. Forced overexpression of cytokine receptors could rescue the JAK2V617F SH2 mutant supporting a critical role of JAK2V617F abundance for constitutive activation. However, under physiologic cytokine receptor expression the SH2 domain is absolutely necessary for oncogenic JAK2V617F activation. This is demonstrated in a bone marrow transplantation model, in which an intact SH2 domain in JAK2V617F is required for the induction of an MPN-like disease. Thus, our results points to an indispensable role of the SH2 domain in JAK2V617F-induced MPNs.

The IL-3, IL-5, and GM-CSF common receptor beta chain mediates oncogenic activity of FLT3-ITD-positive AML.

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.

Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo.

An important limitation of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3-ITD positive AML is the development of resistance. To better understand resistance to FLT3 inhibition, we examined FLT3-ITD positive cell lines which had acquired resistance to midostaurin or sorafenib. In 6 out of 23 TKI resistant cell lines we were able to detect a JAK1 V658F mutation, a mutation that led to reactivation of the CSF2RB-STAT5 pathway. Knockdown of JAK1, or treatment with a JAK inhibitor, resensitized cells to FLT3 inhibition. Out of 136 patients with FLT3-ITD mutated AML and exposed to FLT3 inhibitor, we found seven different JAK family mutations in six of the cases (4.4%), including five bona fide, activating mutations. Except for one patient, the JAK mutations occurred de novo (n = 4) or displayed increasing variant allele frequency after exposure to FLT3 TKI (n = 1). In vitro each of the five activating variants were found to induce resistance to FLT3-ITD inhibition, which was then overcome by dual FLT3/JAK inhibition. In conclusion, our data characterize a novel mechanism of resistance to FLT3-ITD inhibition and may offer a potential therapy, using dual JAK and FLT3 inhibition.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F-myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.