Oncogenic SHP2 Research Articles

In silico mutagenesis-based designing of oncogenic SHP2 peptide to inhibit cancer progression

Cancer is among the top causes of death, accounting for an estimated 9.6 million deaths in 2018, it appeared that approximately 500,000 people die from cancer in the United States alone annually. The SHP2 plays a major role in regulation of cell growth, proliferation, and differentiation, and functional upregulation of this enzyme is linked to oncogenesis and developmental disorders. SHP2 activity has been linked to several cancer types for which no drugs are currently available. In our study, we aimed to design peptide inhibitors against the SHP2 mutant. The crystal structure of the human Src SH2-PQpYEEIPI peptide mutant was downloaded from the protein databank. We generated several peptides from the native wild peptide using an in silico mutagenesis method, which showed that changes (P302W, Y304F, E306Q, and Q303A) might boost the peptide's affinity for binding to SHP2. Furthermore, the dynamical stability and binding affinities of the mutated peptide were confirmed using Molecular dynamics simulation and Molecular Mechanics with Generalized Born and Surface Area Solvation free energy calculations. The proposed substitution greatly enhanced the binding affinity at the residue level, according to a study that decomposed energy into its component residues. Our proposed peptide may prevent the spread of cancer by inhibiting SHP2, according to our detailed analyses of binding affinities.

99 (PB089) - Discovery of an allosteric small molecule inhibitor that can potently target SHP2 in vitro and in vivo

Background: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is the only known tumor-promoting phosphatase, that relays the signals from the diverse receptor tyrosine kinases to the RAS/MAPK pathway. Abnormal SHP2 activation and activating mutations are frequently observed in various cancers and accordingly genetic or pharmacological inhibition of SHP2 has been reported to suppress RTK/KRAS-mediated cancer progression. Moreover, SHP2 serves as a key component of the ERK re-activation in the resistance mechanism to KRAS pathway inhibitors. Therefore, SHP2 inhibitor has a great therapeutic potential in treating RTK/ RAS-mediated cancer as monotherapy and in combination treatment. Material and methods: Biochemical phosphatase assays were performed by detecting DiFMU which is converted by SHP2(WT or mutants) with various concentrations of compounds. For the cellular pERK analysis, compounds were treated into H358 cells followed by pERK measurement using the AlphaLISA system (PerkinElmer). Cell viability was measured by CellTiter-Glo (Promega) after 3-day treatment with compounds. For the in vivo tumor model, cancer cells were inoculated into the flank region of BALB/ c nude mice. Once the tumor size reached 100 mm3, compounds were administrated (P.O.,Q.D). Tumor size was measured twice a week. Results: Cpd1, an orally available and highly potent SHP2 inhibitor, profoundly suppressed SHP2 activity in vitro and KRAS-mutated tumor growth in mouse xenograft models, outcompeting the positive control TNO-155. In detail, Cpd1 potently inhibited SHP2(WT) and also the highly-active oncogenic SHP2 mutants. It impeded the proliferation of cancer cells encoding KRAS and/or SHP2 mutations. With an excellent PK profile, Cpd1 induced in vivo tumor regression in KRAS mutation-driven models. Conclusions: Cpd1 is a potent and selective SHP2 allosteric inhibitor, with a great anti-tumor effect in vitro and in vivo. It may provide a novel therapeutic option for diverse cancers, including KRAS-driven tumors, as a monotherapy or in combination therapies. No conflict of interest.

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain–containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal–regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.

Establishment of Stable Cell Lines Representing Juvenile Myelomonocytic Leukemia with SHP2 Mutations

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. DisclosuresNo relevant conflicts of interest to declare.

Structural reorganization of SHP2 by oncogenic mutations and implications for oncoprotein resistance to allosteric inhibition

Activating mutations in PTPN11, encoding the cytosolic protein tyrosine phosphatase SHP2, result in developmental disorders and act as oncogenic drivers in patients with hematologic cancers. The allosteric inhibitor SHP099 stabilizes the wild-type SHP2 enzyme in an autoinhibited conformation that is itself destabilized by oncogenic mutations. Here, we report the impact of the highly activated and most frequently observed mutation, E76K, on the structure of SHP2, and investigate the effect of E76K and other oncogenic mutations on allosteric inhibition by SHP099. SHP2E76K adopts an open conformation but can be restored to the closed, autoinhibited conformation, near-identical to the unoccupied wild-type enzyme, when complexed with SHP099. SHP099 inhibitory activity against oncogenic SHP2 variants in vitro and in cells scales inversely with the activating strength of the mutation, indicating that either oncoselective or vastly more potent inhibitors will be necessary to suppress oncogenic signaling by the most strongly activating SHP2 mutations in cancer.

Abstract 4879: Mutation selectivity of the allosteric SHP2 inhibitor SHP099

Abstract SHP2 (PTPN11) mediates oncogenic signaling and gain-of-function (GOF) SHP2 mutations are associated with leukemias and other types of cancer. Recently, SHP099 was identified as a novel allosteric SHP2 inhibitor that displayed high potency and selectivity to the wildtype SHP2 in vitro and inhibited protein tyrosine kinase oncogene-driven cancer cells. However, it was unclear whether SHP099 inhibits SHP2 mutants. Using SHP2 mutant-dependent TF-1 leukemia cells, we determined sensitivities of four common oncogenic SHP2 mutants to SHP099. SHP2D61Y-, SHP2A72V-, and SHP2E76K-expressing TF-1 cells were resistant, whereas SHP2E69K-expressing TF-1 cells were sensitive to SHP099 with a potency similar to that of cytokine-stimulated TF-1 cells expressing the wildtype SHP2. Consistently, SHP099 reduced active ERK1/2 and Bcl-XL and induced apoptosis of SHP2E69K-dependent TF-1 cells, but had no effects on the other SHP2 mutant cell lines. SHP2 knockout cells were unresponsive to SHP099, demonstrating specificity. Like the wildtype SHP2, SHP2E69K PTP was inhibited by SHP099 in vitro with a sub-micromolar IC50, whereas micromolar IC50s of SHP099 were observed against SHP2D61Y, SHP2A72V, and SHP2E76K. These results indicate that SHP2D61Y, SHP2A72V, and SHP2E76K mutants are resistant to SHP099 and that SHP2E69K is a SHP099-sensitive oncogenic mutant. Citation Format: Xiaojun Sun, Yuan Ren, Steven Gunawan, Peng Teng, Zhengming Chen, Harshani R. Lawrence, Jianfeng Cai, Nicholas J. Lawrence, Jie Wu. Mutation selectivity of the allosteric SHP2 inhibitor SHP099 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4879.

The Shp2-induced epithelial disorganization defect is reversed by HDAC6 inhibition independent of Cdc42.

Regulation of Shp2, a tyrosine phosphatase, critically influences the development of various diseases. Its role in epithelial lumenogenesis is not clear. Here we show that oncogenic Shp2 dephosphorylates Tuba to decrease Cdc42 activation, leading to the abnormal multi-lumen formation of epithelial cells. HDAC6 suppression reverses oncogenic Shp2-induced multiple apical domains and spindle mis-orientation during division in cysts to acquire normal lumenogenesis. Intriguingly, Cdc42 activity is not restored in this rescued process. We present evidence that simultaneous reduction in myosin II and ERK1/2 activity by HDAC6 inhibition is responsible for the reversion. In HER2-positive breast cancer cells, Shp2 also mediates Cdc42 repression, and HDAC6 inhibition or co-suppression of ERK/myosin II promotes normal epithelial lumen phenotype without increasing Cdc42 activity. Our data suggest a mechanism of epithelial disorganization by Shp2 deregulation, and reveal the cellular context where HDAC6 suppression is capable of establishing normal epithelial lumenogenesis independent of Cdc42.

Shp2 promotes metastasis of prostate cancer by attenuating the PAR3/PAR6/aPKC polarity protein complex and enhancing epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT), marked by the dissolution of cell-cell junctions, loss of cell polarity and increased cell motility, is one of the essential steps for prostate cancer metastasis. However, the underlying mechanism has not been fully explored. We report in this study that Shp2 is upregulated in prostate cancers and is associated with a poor disease outcome, namely tumor metastasis and shortened patient survival. Overexpression of wild-type Shp2 or an oncogenic Shp2 mutant leads to increased prostate cancer cell proliferation, colony and sphere formation, and in vivo tumor formation. Opposite effects are seen in Shp2-knockdown cells. Moreover, Shp2 promotes in vitro migration and in vivo metastasis of prostatic tumor cells. Mechanistically, Shp2 interacts with PAR3 (partitioning-defective 3) via its Src homology-2 domain. Ectopic expression of Shp2 attenuates the phosphorylation of PAR3 and the formation of the PAR3/PAR6/atypical protein kinase C polarity protein complex, resulting in disrupted cell polarity, dysregulated cell-cell junctions and increased EMT. These findings provide a novel mechanism by which oncogenic signal-transduction molecules regulate cell polarity and induction of EMT.

Therapeutic potential of targeting the oncogenic SHP2 phosphatase.

The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase associated with various kinds of leukemia and solid tumors. Thus, there is substantial interest in developing SHP2 inhibitors as potential anticancer and antileukemia agents. Using a structure-guided and fragment-based library approach, we identified a novel hydroxyindole carboxylic acid-based SHP2 inhibitor 11a-1, with an IC50 value of 200 nM and greater than 5-fold selectivity against 20 mammalian PTPs. Structural and modeling studies reveal that the hydroxyindole carboxylic acid anchors the inhibitor to the SHP2 active site, while interactions of the oxalamide linker and the phenylthiophene tail with residues in the β5–β6 loop contribute to 11a-1’s binding potency and selectivity. Evidence suggests that 11a-1 specifically attenuates the SHP2-dependent signaling inside the cell. Moreover, 11a-1 blocks growth factor mediated Erk1/2 and Akt activation and exhibits excellent antiproliferative activity in lung cancer and breast cancer as well as leukemia cell lines.

Oncogenic Shp2 disturbs microtubule regulation to cause HDAC6-dependent ERK hyperactivation

Deregulation of Shp2, a non-receptor tyrosine phosphatase, causes hyperactivation of extracellular signal-regulated kinase (ERK), leading to growth abnormality. Here, we show that inhibition of RhoA-Dia is sufficient to upregulate ERK activation in epithelial cells. Oncogenic Shp2 expression attenuates RhoA-Dia signaling, by which microtubule (MT) is destabilized with reduced level of acetylation. Either MT stabilization, silencing of histone deacetylase 6 (HDAC6) or enforcing RhoA-Dia signal prevents oncogenic Shp2-induced ERK hyperactivation. We provide evidence that downregulation of RhoA-Dia-EB1 pathway by oncogenic Shp2 leads to HDAC6-mediated reduction in MT acetylation, in turn affecting ERK regulation. In response to serum stimulation, cells expressing wild-type Shp2 display transient ERK activation. In contrast, cells expressing oncogenic Shp2 have prolonged ERK activation. HDAC6 inhibition diminishes sustained activation of ERK and slows down the growth of these cells. Likewise, in human cancer cells, blocking Shp2 increases MT acetylation and decreases ERK phosphorylation, which are reversed by inhibition of Dia. As such, HDAC6 inhibition in these cells also reduces ERK activity. Our findings link MT regulation by HDAC6 to oncogenic Shp2 and ERK regulation, implicating the therapeutic potential of HDAC6 inhibitor in diseases involving Shp2 deregulation.

Down-Modulation of Shp1 and Hsp70 Provides a Survival Advantage to the Ph+ Cells of CML Patients Additive to That Related to the Oncogenic BCR/ABL Protein and May Account for Resistance to Ima Treatment.

Some chronic myeloid leukemia (CML) patients in chronic-phase and even more in advanced-phase, demonstrate primary resistance to imatinib or develop secondary resistance during treatment. In this study we investigate the mechanisms of resistance in a couple of Ima-sensitive and -resistant Kcl22 cell lines (Kcl22-s and Kcl22-r) combining a gene expression profile and proteomics approach. This couple of cells is interesting for a series of reasons: none of the known mechanisms of Ima resistance, i.e. Bcr-Abl point mutations or its overexpression or MDR up-regulation, has been described in these cells; in addition, differently respect to the various Ph+ cell lines and more similarly to a Ph+ stem cells, the original parental Kcl22-s show a high level of resistance to the Ima-induced apoptosis. Microarray indicated that 153 genes are up-regulated and 103 are down-regulated in the Kcl22-r respect to the Kcl22-s cells. Up-regulated genes comprise genes encoding for some membrane transporters, cell signalling proteins including MAPK kinase pathway molecules, cancer testis antigens, apoptosis negative regulators and DNA repair proteins. Among the down-regulated genes, that comprised also Annexin-1 and the HSP-70 and HSP27 proteins, we found a very low levels of the non-receptor, SH2 domain-containing protein tyrosin phosphatase-1 (Shp1). This protein is widely expressed in hematopoietic cells, and is directly involved in the negative regulation of cytokine/protein tyrosine kinase-mediated signalling. Methylation-specific PCR analysis of specific regions of SHP-1 and HSP70 promoters showed that the down-regulation of these genes is related to the hypermethylation of their promoters. Treatment of Kcl22-r with the 5-Azacytidine (5-AC), a demethylant agent, resulted in induction of Shp1 thus providing a further confirmation for the role of promoter methylation in the down regulation of this gene. Interestingly, the 5-AC-induced expression of Shp1 correlated, in Kcl22-r, with a significant reduction of phosphorylation of both pSTAT3 and pERK1/2, as well as of proliferation capacity of these cells. The phosphorylation of these latter signalling proteins is rather low in Kcl22-s cells independently by the Bcr-Abl p210 activity and it is not changed by the 5-AC treatment. We also show that Shp1 interacts in these cells with the Shp2, another SH2-containing phosphatase with different biological properties. Indeed, it has been shown that Shp2 is able to positively regulate signalling pathways, including the Ras/MAPK pathway. Moreover, the Shp2-dependent activation of the Ras/MAPK pathway in Kcl22-r cells may be also due to Hsp70 protein down-regulation. Indeed, this chaperone protein has been recently shown to stabilize the inactive form of the oncogenic Shp2 phosphatase. Taken together, our results indicate that a complex pathway of proliferation and survival in Ph+ cells based on the balance between the levels of Shp1 and 2 phosphatases and Hsp70 may account for the Bcr-Abl independent activation of Ras/MAPK pathway and for the resistance to Ima.

Shp-2 heterozygous hematopoietic stem cells have deficient repopulating ability due to diminished self-renewal

Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function. Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies. Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells. These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.

Shp-2 Heterozygous Hematopoietic Stem Cells Have Deficient Repopulating Activity Due to a Self-Renewal Defect.

The protein tyrosine phosphatase, Shp-2, has been shown to be necessary for normal hematopoiesis based on embryonic stem (ES) cell-based assays; however, due to the early lethality of the homozygous Shp-2 mutant mice (Shp-2−/−) the role of Shp-2 in adult hematopoietic stem cell (HSC) function has never been examined. The Shp-2 heterozygous mice (Shp-2+/−) bear a mutant allele of the Shp-2 gene resulting in the production of a mutant protein lacking amino acids 46–110, which confers a loss of function. To test the hypothesis that Shp-2 is required for normal HSC activity, we compared the competitive repopulating ability of Shp-2+/− bone marrow-derived cells with WT cells. Total adult bone marrow low density mononuclear cells were isolated from Shp-2+/− and WT littermate controls (test cells, C57Bl/6 background, CD45.2+), mixed with a common pool of competitor (comp) cells (BoyJ background, CD45.1+), and administered to lethally irradiated (1100 cGy split dose) Gpi/BoyJ recipients. Based on peripheral blood chimerism, the repopulating ability of the Shp-2+/− cells was significantly lower than that of the WT cells (Figure 1, *p<0.0001 Shp-2+/− v. WT at ratio 1:2; **p=0.001 Shp-2+/− v. WT at ratio 1:1). We next converted the chimerism to repopulating units using the formula [competitor number x 105] X [% 45.2]/100 − [% 45.2] to quantitatively asses the repopulating defect in Shp-2+/− HSCs. We observed that the repopulating units of the Shp-2+/− cells was approximately 3-fold lower than that of the WT cells at both cell doses administered (Figure 2, *p=0.003 Shp-2+/− v. WT at ratio 1:2; **p=0.03 comparing Shp-2+/− v. WT at ratio 1:1). Multi-lineage analysis using two color fluorescence cytometry revealed a significantly lower contribution of Shp-2+/− cells to all lineages tested (B220, GR1, Mac, and CD4/8) compared to WT cells. [Display omitted] As Shp-2 has been shown to participate in cell migration, we sought to rule out a homing deficiency of the Shp-2+/− HSCs. We performed short term homing assays and observed no difference in spleen-homed or bone marrow-homed Shp-2+/− and WT lin- cells twenty hours following transplantation. To evaluate self-renewal potential, we conducted serial transplantation experiments. Total bone marrow low density mononuclear cells were isolated from primary or seconary recipient mice with equal chimerism and transplanted into lethally irradiated (1100 cGy split dose) Gpi/BoyJ recipients. While no significant difference was observed between Shp-2+/− and WT engraftement in secondary transplants, eight weeks following tertiary transplantation, engraftment of the Shp-2+/− cells is significantly lower than that of the WT cells (WT 68.9% +/− 9.5 v. Shp-2+/− 26.1% +/− 11.7, n=6, p<0.0001) suggesting that a self-renewal defect contributes to the decreased HSC activity of the Shp-2+/− cells. These data demonstrate that Shp-2 function is not only necessary within the progenitor compartment to support proficient hematopoiesis, but is also needed within the HSC compartment to support normal HSC self-renewal. These findings provide insight into how oncogenic Shp-2 potentially may contribute to the dysregulation of hematopoiesis and the pathogenesis of childhood leukemias.