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Frequently mutated in Acute myeloid leukemia (AML), FLT3 is considered as one of the favorable targets for treatment. The FLT3 internal tandem duplication (ITD) mutation enhances kinase activity and causes hyperactivation of downstream signal transduction. Several small molecule FLT3 inhibitors have developed, but their clinical efficacy is limited due to generation of drug resistance. In this study, we define a new mechanism of drug resistance toward tyrosine kinase inhibitors (TKIs). Initially, we found a rapid decrease in the protein level of tumor suppressor p53 in FLT3-ITD-positive MV4-11 and MOLM13 cells and peripheral blood mononuclear cells (PBMCs) from FLT3-ITD AML patients upon treatment with TKIs including sorafenib, sunitinib and quizartinib. The decrease is not caused by changes in mRNA expression as revealed by qPCR analyses but rather by accelerated protease degradation because the p53 protein was stabilized by proteasome inhibitor MG132. Furthermore, treatment of cells with RG7388, a potent disruptor of p53 and MDM2 interaction, prevented the TKI-induced p53 loss. Since MDM2 is the most important E3 ligase responsible for ubiquitination of p53, the data suggest that TKIs may lead to the degradation of p53 by promoting ubiquitination. Indeed, ubiquitination assays verified that TKIs promoted K48 poly-ubiquitination of p53. Previous studies have demonstrated that activations of FLT3 downstream signaling components such as ERKs and Akt reduce p53 protein stability through ubiquitination by activating MDM2. It is somewhat unexpected that inhibition of FLT3-ITD and its downstream signaling pathways also resulted in decreased p53 stability due to increased ubiquitination. We treated FLT3-ITD-containing cells with specific ERK, AKT and STAT5 inhibitors. Interestingly, while inhibition of ERKs and AKT had no significant effect on the stability of p53, STAT5 inhibition resulted in a reduced level of p53 accompanied by increased K48 poly-ubiquitination. We further analyzed the interaction of p53 with MDM2 in AML cells by using immunoprecipitation. The results showed that the p53-MDM2 interaction was significantly enhanced after treatment with TKIs and STAT5 inhibitors, which was diminished in the presence of RG7388. Subcellular fractionation revealed the presence of p53 and STAT5 in both nucleus and cytoplasm. Treatment of cells with TKIs resulted in a decreased level of p53 and STAT5 in the nucleus, and immunoprecipitation of nuclear proteins with a p53 antibody revealed a reduced association of p53 with STAT5. Taken together, the data suggest that FLT3 inhibitors inhibited nuclear translocation of STAT5 and reduced its interaction of p53 thereby facilitating p53/MDM2 interaction and subsequent ubiquitination and degradation of p53. This study reveals a novel mechanism by which drug resistance to TKIs may occur and further support the use of MDM2/p53 interaction inhibitors in combination with TKIs for treatment of AML. Disclosures No relevant conflicts of interest to declare.

Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm featured by impaired differentiation and uncontrolled proliferation of myeloid progenitors. Gain-of-function mutations of FMS-like tyrosine kinase 3 (FLT3) present in 30-40% of patients with AML. In addition, more than 90% of AML blasts aberrantly express FLT3, making FLT3 an attractive therapeutic target for AML. Currently, several small molecule tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved in the treatment of AML, but they need to be used in combination with chemotherapy because of their limited potency to eliminate leukemic cells as single agents, largely due to the development of secondary inhibitor-resistant FLT3 mutations. Therefore, novel therapeutic strategies targeting FLT3 are needed. In the present study, we developed a FLT3 ligand-emtansine drug conjugate (FL-DM1) that targeted FLT3-positive AML cells with high potency and selectivity. We expressed recombinant human FLT3 ligand (rhFL) in the periplasm of recombinant E. Coli. The protein was purified by a two-step purification system containing Ni-NTA and Phenyl Sepharose. Our purified rhFL was bioactive to stimulate phosphorylation of FLT3 and proliferation of THP-1 cells. Next, we conjugated purified rhFL and emtansine (DM1) with SPDP linker. Reducing and non-reducing SDS-PAGE revealed that rhFL and DM1 were successfully conjugated, evidenced by a band with higher molecular weight of the conjugation product. Previous studies show that DM1 is a drug preferentially targeting proliferating cells by depolymerizing microtubules through binding at the vinca binding site of tubulin. We found that FL-DM1 reserved the physiological function of FLT3 ligand to stimulate proliferation of AML cells by inducing phosphorylation of FLT3 and the downstream signaling protein AKT in immunoblot, potentially enhancing the potency of FL-DM1 to inhibit FLT3-positive AML cells. Furthermore, flow cytometry showed that the surface expression of FLT3 significantly decreased on cells treated by FL-DM1 within two hours, which indicated the internalization of FL-DM1/FLT3 complex on these FLT3-positive AML cells, providing a mechanism of FL-DM1 entering target cells. In cell viability assay, we found that FL-DM1 effectively inhibited FLT3-positive AML cells THP-1 and MV-4-11 with IC50 around 10-30 nM. Also, FL-DM1 induced apoptosis and cell cycle arrest at G2/M phase in these cells detected by flow cytometry. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are FLT3-negative erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. We found that FL-DM1 inhibited HCD-57 cells transformed by FLT3-ITD, but not parental HCD-57 cells without FLT3 expression, indicating the selectivity of FL-DM1 to target FLT3-positive AML cells. In conclusion, our data demonstrated that FL-based drug conjugate can serve as an effective drug to target FLT3-expressing AML cells. Further studies will focus on in-vivo evaluation of FL-DM1 in animal models, the production of uncleavable SMCC linked FL-DM1 with improved in-vivo pharmacokinetic properties, and screening of FL muteins-DM1 conjugates with desired pharmacological properties. Disclosures No relevant conflicts of interest to declare.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of B lymphocytes blocked at an early stage of differentiation, which accounts for approximately 30% of childhood cancers. Almost all B-ALL blasts aberrantly express FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase involved in the development of dendritic cells and B lymphocytes. In B-ALL, the high expression of FLT3 in blasts significantly increases the odds of relapse/death in patients, indicating a pathological role of FLT3 in B-ALL. Note that gain-of-function mutations of FLT3 are relatively rare ( Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a hematological malignancy featured by impaired differentiation and uncontrolled proliferation of myeloid blasts. FLT3 internal tandem duplication (ITD) presents in 30-40% patients with AML, which serves as an independent poor prognostic marker and an attractive therapeutic target. Up to date, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved by FDA in the treatment of AML. However, FLT3 TKIs as single agents have limited effects to eliminate AML cells due to multiple drug-resistant mechanisms, including secondary FLT3 mutations, alternatively activated cell survival pathways, upregulation of FLT3 ligand, and downregulation of tumor suppressor genes. In the present study, we found that FLT3 TKIs decreased tumor suppressor p53 protein level by downregulation of YOD1 through miR-181 in FLT3-ITD mutant AML cells. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. By using transcriptome analysis with RNAseq, we identified multiple differentially expressed miRNAs in HCD-57 transformed by FLT3-ITD, compared with parental HCD-57 cells. miR-181a-5p and miR-181b-5p were among these highly differentially expressed miRNAs. These two miRNAs were predicted to bind in 3'UTR of deubiquitinase YOD1 by using TargetScanHuman 7.2, an online tool to predict biological targets of miRNAs. Previous studies have shown that the ubiquitination and protein level of p53 is dysregulated due to overexpressed E3 ligase MDM2/4 in AML cells, but few studies focused on deubiquitinase of p53. We found that deubiquitinase YOD1 interacted with p53 by immunoprecipitation. Overexpression of YOD1 prevented degradation of p53 led by cycloheximide, a protein synthesis inhibitor. We further found that overexpression of YOD1 resulted in decreased ubiquitination of p53, indicating that YOD1 stabilized p53 protein through deubiquitination. Subsequently, we detected the expression of miR-181, YOD1 and p53 in a FLT3-ITD positive AML cell line MV-4-11 treated by FLT3 TKIs sorafenib, sunitinib and quizartinib. Real-time quantitative PCR showed that the treatment of FLT3 TKIs upregulated the expression of miR-181a-5p/miR-181b-5p, and downregulated mRNA level of their predicted target YOD1. The mRNA level of p53 remained unchanged but its protein level decreased with enhanced ubiquitination in MV-4-11 cells treated by FLT3 TKIs in the ubiquitination assay. These data suggested that FLT3 TKIs could reduce the stability of p53 by regulating miRNA-targeted YOD1. In addition, we collected peripheral blood mononuclear cells (PBMCs) from patients with AML and age-matched healthy donors. We found increased expression of miR-181a-5p and miR-181b-5p and decreased expression of YOD1 in PBMCs from AML patients compared with healthy donors, suggesting a pathological role of miR-181 to regulate YOD1/53 pathway in AML. In conclusion, our data showed that FLT3 TKIs induced instability of p53 by miR-181 mediated downregulation of YOD1. YOD1 as a novel deubiquitinase of p53 could play important roles in drug-resistance and progression of AML. Disclosures No relevant conflicts of interest to declare.