Rett syndrome (RTT) is a severe neurodevelopmental disorder in which current therapeutic strategies remain largely focused on providing symptomatic relief without addressing underlying disease mechanisms. In contrast, we have identified the protein tyrosine phosphatase PTP1B as a mechanism-based therapeutic target and evaluated a class of selective, allosteric small-molecule inhibitors in female murine models of RTT. We show that one of these compounds localizes to brain regions central to motor coordination and cardio-respiratory control, which are core domains of RTT pathology. Pharmacological inhibition of PTP1B produces robust and sustained improvement in multiple disease symptoms, including muscle weakness, motor and coordination deficits, and cardiac and respiratory dysfunction. Concordant results obtained with genetic ablation of PTP1B, with effects maintained for over one year, demonstrate that phenotypic rescue arises from on-target modulation of disease-relevant signaling. Mechanistically, PTP1B inhibition is known to normalize neurotrophic and metabolic pathways, including TRKB and insulin/leptin signaling, thereby restoring circuit-level function. These findings establish PTP1B as a clinically actionable, disease-modifying target and demonstrate that selective, allosteric inhibition of a protein tyrosine phosphatase can achieve durable therapeutic benefit in vivo. This work provides a strong rationale for the clinical evaluation of PTP1B inhibitors as a mechanism-based treatment strategy for RTT. One Sentence Summary:We have validated inhibition of PTP1B as a mechanism-based therapeutic strategy that alleviates a wide range of symptoms in a mouse model of Rett syndrome.

Background/Objectives: The recent development of four-dimensional X-ray velocimetry (4DXV) technology (three-dimensional space and time) provides a unique opportunity to obtain preclinical quantitative functional lung images. Only single-scan measurements in non-survival studies have been obtained to date; thus, methodologies enabling animal survival for repeated imaging to be accomplished over weeks or months from the same animal would establish new opportunities for the assessment of pathophysiology drivers and treatment response in advanced preclinical drug-screening efforts. Methods: An anesthesia protocol developed for animal recovery to allow for repetitive, longitudinal scanning of individual animals over time. Test–retest imaging scans from the lungs of healthy mice were performed over 8 weeks to assess the repeatability of scanner-derived quantitative imaging metrics and variability. Results: Using a murine model of fibroproliferative lung disease, this longitudinal scanning approach captured heterogeneous progressive changes in pulmonary function, enabling the visualization and quantitative measurement of averaged whole lung metrics and spatial/regional change. Radiation dosimetry studies evaluated the effects of imaging acquisition protocols on X-ray dosage to further adapt protocols for the minimization of radiation exposure during repeat imaging sessions using these newly developed image acquisition protocols. Conclusions: Overall, we have demonstrated that the 4DXV advanced imaging scanner allows for repeat measurements from the same animal over time to enable the high-resolution, noninvasive mapping of quantitative lung airflow dysfunction in mouse models with heterogeneous pulmonary disease. The animal anesthesia and image acquisition protocols described will serve as the foundation on which further applications of the 4DXV technology can be used to study a diverse array of murine pulmonary disease models. Together, 4DXV provides a novel and significant advancement for the longitudinal, noninvasive interrogation of pulmonary disease to assess spatial/regional disease initiation, progression, and response to therapeutic interventions.

The mitogen-activated protein kinase (MAPK) and mechanistic target of rapamycin (mTOR) signaling nodes play a crucial role in many human cancers. Due to the molecular reciprocity between MAPK and mTOR signaling nodes, development of compounds with multikinase targeting was explored. A series of mTOR inhibitor analogs of AZD8055 and AZD2014 were designed to allow for covalent linking to a potent MAPK kinase (MEK) inhibitor to produce a single, bivalent chemical entity. Dual-acting agents (i.e., compound LP-65) were synthesized displaying high in vitro inhibition of both MEK (IC50 = 83.2 nM) and mTOR (IC50 = 40.5 nM). Additionally, compound LP-65 demonstrated significant modulation of MEK and mTOR signaling activity in human glioma cells (D54) and human melanoma cells (A375), with a corresponding decrease in cellular proliferation and migration. Treatment of mice with LP-65 (40 mg/kg) having a myeloproliferative neoplasm, myelofibrosis, revealed down modulation of in vivo signaling pathways and therapeutic efficacy.

The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate complexes during lysis and enrichment procedures. Advantages of this new approach are illustrated through application to PTP1B interaction networks in models of HER2-positive and Herceptin-resistant breast cancer. We have demonstrated that inhibitors of PTP1B significantly reduced proliferation and viability in cell-based models of acquired and de novo Herceptin resistance in HER2-positive breast cancer. Using differential analysis, comparing substrate-trapping to wild-type PTP1B, we have identified multiple unreported protein targets of PTP1B with established links to HER2-induced signaling and provided internal validation of method specificity through overlap with previously identified substrate candidates. Overall, this versatile approach can be readily integrated with evolving proximity-labeling platforms (TurboID, BioID2, etc.), and is broadly applicable across all PTP family members for the identification of conditional substrate specificities and signaling nodes in models of human disease.

Quantitative MRI biomarkers are sought to replace painful and invasive sequential bone-marrow biopsies routinely used for myelofibrosis (MF) cancer monitoring and treatment assessment. Repeatability of MRI-based quantitative imaging biomarker (QIB) measurements was investigated for apparent diffusion coefficient (ADC), proton density fat fraction (PDFF), and magnetization transfer ratio (MTR) in a JAK2 V617F hematopoietic transplant model of MF. Repeatability coefficients (RCs) were determined for three defined tibia bone-marrow sections (2–9 mm; 10–12 mm; and 12.5–13.5 mm from the knee joint) across 15 diseased mice from 20–37 test-retest pairs. Scans were performed on consecutive days every two weeks for a period of 10 weeks starting 3–4 weeks after transplant. The mean RC with (95% confidence interval (CI)) for these sections, respectively, were for ADC: 0.037 (0.031, 0.050), 0.087 (0.069, 0.116), and 0.030 (0.022, 0.044) μm2/ms; for PDFF: 1.6 (1.3, 2.0), 15.5 (12.5, 20.2), and 25.5 (12.0, 33.0)%; and for MTR: 0.16 (0.14, 0.19), 0.11 (0.09, 0.15), and 0.09 (0.08, 0.15). Change-trend analysis of these QIBs identified a dynamic section within the mid-tibial bone marrow in which confident changes (exceeding RC) could be observed after a four-week interval between scans across all measured MRI-based QIBs. Our results demonstrate the capability to derive quantitative imaging metrics from mouse tibia bone marrow for monitoring significant longitudinal MF changes.

Cancer signaling pathways can readily adapt to evade therapy through intrinsic resistance or compensatory mechanisms driving a resistant state. The prominence of oncogenic kinase signaling in a multitude of cancer types has inspired development of molecularly targeted drugs, facilitating combination treatment strategies designed to overcome adaptations such as signaling crosstalk and activation of downstream effectors. Treatments using inhibitor combinations, however, remain a challenge as clinical trials have been fraught with dose-limiting toxicities. Avoidance of hepatic first-pass metabolism by transport and sequestration into mesenteric gastrointestinal lymphatic vessels may improve drug exposure and reduce dose-limiting toxicities often observed in combination therapy studies. Despite potential advantages, lymphatically directed kinase inhibitors have remained unexplored due in part to limited understanding of the physicochemical properties required for lymphatic uptake of small molecules. We demonstrate a first-in-class series of orally bioavailable small molecule kinase inhibitors which are intrinsically ‘lymphatropic’, undergoing lymphatic absorption to achieve sustained circulatory drug levels and therapeutic benefit, with no observable toxicity. Single agent multifunctional inhibitors of high-value targets (MEK/PI3K, MEK/PI3K/mTOR, & MEK/mTOR) were designed and synthesized. Compounds had unique physiochemical properties, demonstrating high lymphatic uptake in a mesenteric lymphatic vessel cannulation model and in isolated lymph nodes extracts. Pharmacokinetic studies in mice following oral administration revealed that sustained plasma blood levels were achieved within the therapeutic window for up to 24 hours. Oral bioavailability in mice was found to be 80-85% of drug administered and was constant over a single-dose range study (100 to 1,000 mg/kg). On-target in vitro and in vivo inhibition of activated MEK and PI3K was also demonstrated, revealing high potency of both the parent molecule and therapeutically active metabolites. Together, these studies revealed that lymphatic sequestration of orally dosed compounds serves to provide a reservoir that circumvents pharmacotoxicity through controlled physiological drug release into the systemic circulation and avoidance of first-pass hepatic metabolism. This platform provides a template for further development of additional lymphatropic small molecule inhibitors presenting opportunities in which multi-targeted cancer treatment options can now be evaluated. Exciting new therapeutic prospects offered by lymphatropic agent development is anticipated to transform treatment options for a wide variety of cancer types such as metastasis and lymphoma along with a myriad of other diseases including autoimmune disorders. Citation Format: Brian D. Ross, Youngsoon Jang, Amanda Welton, Christopher A. Bonham, Kevin Heist, Lucas McDonald, Gary D. Luker, Thomas L. Chenevert, Marcian Van Dort. Orally bioavailable 'lymphatropic' kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3936.

Activation of compensatory signaling nodes in cancer often requires combination therapies that are frequently plagued by dose-limiting toxicities. Intestinal lymphatic drug absorption is seldom explored, although reduced toxicity and sustained drug levels would be anticipated to improve systemic bioavailability. A potent orally bioavailable multi-functional kinase inhibitor (LP-182) is described with intrinsic lymphatic partitioning for the combined targeting of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways without observable toxicity. We demonstrate selectivity and therapeutic efficacy through reduction of downstream kinase activation, amelioration of disease phenotypes, and improved survival in animal models of myelofibrosis. Our further characterization of synthetic and physiochemical properties for small molecule lymphatic uptake will support continued advancements in lymphatropic therapy for altering disease trajectories of a myriad of human disease indications.

Yeast VH1-related phosphatase (YVH1) (also known as DUSP12) is a member of the atypical dual-specificity phosphatase subfamily. Although no direct substrate has been firmly established, human YVH1 (hYVH1) has been shown to protect cells from cellular stressors, regulate the cell cycle, disassemble stress granules, and act as a 60S ribosome biogenesis factor. Despite knowledge of hYVH1 function, further research is needed to uncover mechanisms of its regulation. In this study, we investigate cellular effects of a Src-mediated phosphorylation site at Tyr(179) on hYVH1. We observed that this phosphorylation event attenuates localization of hYVH1 to stress granules, enhances shuttling of hYVH1 to the nucleus, and promotes hYVH1 partitioning to the 60S ribosomal subunit. Quantitative proteomics reveal that Src coexpression with hYVH1 reduces formation of ribosomal species that represent stalled intermediates through the alteration of associating factors that mediate translational repression. Collectively, these results implicate hYVH1 as a novel Src substrate and provide the first demonstrated role of tyrosine phosphorylation regulating the activity of a YVH1 ortholog. Moreover, the ribosome proteome alterations point to a collaborative function of hYVH1 and Src in maintaining translational fitness.

Myelofibrosis is a myeloproliferative neoplasm (MPN) caused primarily by mutations in hematopoietic stem and progenitor cells (HSPCs) that activate Janus kinase 2 (JAK2). Central roles for JAK2 signaling in myelofibrosis (MF) have been established, leading to marked extramedullary hematopoiesis, splenomegaly, and bone marrow fibrosis, but the use of JAK inhibitors has shown limited ability to produce durable remissions in most patient populations. Bypass or resistance mechanisms to JAK inhibition have been linked to concomitant activation of compensatory phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways, and while genetic and functional studies have not fully defined the mechanisms for transformation and maintenance of the proliferative state in MF, targeting multiple signaling pathways will be required to alter the disease course and improve therapeutic outcomes. Treatments using kinase inhibitor combinations remain a challenge clinically, and trials have struggled to create positive balance between gains in survival, therapeutic efficacy, and dose-limiting toxicity. A multi-functional kinase inhibitor was developed to deliver consistent and synergistic dosing ratios against multiple targets simultaneously, thereby blocking compensatory oncogenic signaling pathways while minimizing the prospect for adverse effects. Using synthetic medicinal chemistry, we present a potent and selective, orally bioavailable, single-molecule multi-targeted kinase inhibitor (LP-182) against MAPK and PI3K/mTOR signaling pathways. We demonstrate selectivity and therapeutic efficacy through in vitro screening of kinome inhibition, reduction of cell growth and downstream kinase activation, renormalization of immune cell populations, amelioration of splenomegaly and bone marrow fibrosis, and improved survival in the myeloproliferative leukemia oncogene (MPLW515L) mouse model of myelofibrosis. Furthermore, treatment with LP-182 was well tolerated showing no observable pharmacotoxicity, and combination with the JAK inhibitor Ruxolitinib also showed ~35% reduction in spleen size as compared to either treatment alone with a ~60% spleen volume reduction compared to vehicle treatment, in the JAK2+/V617F mutant mouse model. Overall, these data suggest that in vivo bioavailability and distribution of LP-182 to secondary lymphoid tissues attenuates MAPK and PI3K signaling to alleviate disease phenotypes in animal models of MF. Simultaneous targeting of both MAPK and PI3K pathways with LP-182 alone or in combination with JAK inhibition provides the potential for significant new opportunities to improve the clinical outcome of MF patients. Citation Format: Christopher A. Bonham, Youngsoon Jang, Kevin Heist, Amanda Welton, Tanner Robison, Kathryn E. Luker, Gary D. Luker, Winston Y. Lee, Thomas L. Chenevert, Marcian Van Dort, Brian D. Ross. A novel small molecule inhibitor of MAPK and PI3K ameliorates disease phenotypes in myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6366.

Established roles for PI3K and MAPK signaling pathways in tumorigenesis has prompted extensive research towards the discovery of small-molecule inhibitors as cancer therapeutics. However, significant compensatory regulation exists between these two signaling cascades, leading to redundancy among survival pathways. Consequently, initial clinical trials aimed at either PI3K or MEK inhibition alone have proven ineffective and highlight the need for development of targeted and innovative therapeutic combination strategies. We designed a series of PI3K inhibitor derivatives wherein a single morpholine group of the PI3K inhibitor ZSTK474 was substituted with a variety of 2-aminoethyl functional groups. Analogs with pendant hydroxyl or methoxy groups maintained low nanomolar inhibition towards PI3Kα, PI3Kγ, and PI3Kδ isoforms in contrast to those with pendant amino groups which were significantly less inhibitory. Synthesis of prototype PI3K/MEK bifunctional inhibitors (6r, 6s) was guided by the structure-activity data, where a MEK-targeting inhibitor was tethered directly via a short PEG linker to the triazine core of the PI3K inhibitor analogs. These compounds (6r, 6s) displayed nanomolar inhibition towards PI3Kα, δ, and MEK (IC50 ∼105-350 nM), and low micromolar inhibition for PI3Kβ and PI3Kγ (IC50 ∼1.5-3.9 μM) in enzymatic inhibition assays. Cell viability assays demonstrated superior anti-proliferative activity for 6s over 6r in three tumor-derived cell lines (A375, D54, SET-2), which correlated with inhibition of downstream AKT and ERK1/2 phosphorylation. Compounds 6r and 6s also demonstrated in vivo tolerability with therapeutic efficacy through reduction of kinase activation and amelioration of disease phenotypes in the JAK2V617F mutant myelofibrosis mouse cancer model. Taken together, these results support further structure optimization of 6r and 6s as promising leads for combination therapy in human cancer as a new class of PI3K/MEK bifunctional inhibitors.

The protein tyrosine phosphatase PTP1B is a major regulator of glucose homeostasis and energy metabolism, and a validated target for therapeutic intervention in diabetes and obesity. Nevertheless, it is a challenging target for inhibitor development. Previously, we generated a recombinant antibody (scFv45) that recognizes selectively the oxidized, inactive conformation of PTP1B. Here, we provide a molecular basis for its interaction with reversibly oxidized PTP1B. Furthermore, we have identified a small molecule inhibitor that mimics the effects of scFv45. Our data provide proof-of-concept that stabilization of PTP1B in an inactive, oxidized conformation by small molecules can promote insulin and leptin signaling. This work illustrates a novel paradigm for inhibiting the signaling function of PTP1B that may be exploited for therapeutic intervention in diabetes and obesity.

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.

A central feature of the protein tyrosine phosphatase (PTP) catalytic mechanism is an attack of the substrate’s phosphate moiety by a thiolate ion in the signature CX5R motif. In addition to being an effective nucleophile in this form, the thiolate ion is also susceptible to reversible redox regulation. This attribute permits temporal inhibition of PTP activities, which affects numerous cellular processes utilizing kinase-mediated signal propagation. Accumulating evidence has revealed diverse mechanisms adopted by PTPs to avoid irreversible thiol oxidation of the active site Cys residue, often involving structurally proximal thiols within the active site region. Therefore, there has been a significant effort made to develop thiol labeling strategies coupled to mass spectrometry to identify and characterize redox sensitive thiols within PTPs as a necessary step in understanding how a particular PTP is regulated by redox signaling. A common drawback to many current methods is the use of neutral pH labeling techniques, requiring special attention with regards to non-specific thiol oxidation during sample preparation. This study describes the use of rapid, low pH thiol labeling methods to overcome this issue. Mercury immobilized metal affinity chromatography (Hg-IMAC) demonstrated high selectivity and specificity while enriching for thiol-containing peptides from the atypical dual specificity phosphatase hYVH1 (also known as DUSP12). This approach revealed several reversibly oxidized thiols within the catalytic domain of hYVH1. Subsequently, use of another low pH labeling reagent, 4,4-dithiopyridine (4-DTP) helped identify novel disulfide linkages providing evidence that hYVH1 utilizes a disulfide exchange mechanism to prevent irreversible oxidation of the catalytic Cys residue in the active site.

Myotubularin-related 2 (MTMR2) is a 3-phosphoinositide lipid phosphatase with specificity towards the D-3 position of phosphoinositol 3-phosphate [PI(3)P] and phosphoinositol 3,5-bisphosphate lipids enriched on endosomal structures. Recently, we have shown that phosphorylation of MTMR2 on Ser58 is responsible for its cytoplasmic sequestration and that a phosphorylation-deficient variant (S58A) targets MTMR2 to Rab5-positive endosomes resulting in PI(3)P depletion and an increase in endosomal signaling, including a significant increase in ERK1/2 activation. Using in vitro kinase assays, cellular MAPK inhibitors, siRNA knockdown and a phosphospecific-Ser58 antibody, we now provide evidence that ERK1/2 is the kinase responsible for phosphorylating MTMR2 at position Ser58, which suggests that the endosomal targeting of MTMR2 is regulated through an ERK1/2 negative feedback mechanism. Surprisingly, treatment with multiple MAPK inhibitors resulted in a MTMR2 localization shift from Rab5-positive endosomes to the more proximal APPL1-positive endosomes. This MTMR2 localization shift was recapitulated when a double phosphorylation-deficient mutant (MTMR2 S58A/S631A) was characterized. Moreover, expression of this double phosphorylation-deficient MTMR2 variant led to a more sustained and pronounced increase in ERK1/2 activation compared with MTMR2 S58A. Further analysis of combinatorial phospho-mimetic mutants demonstrated that it is the phosphorylation status of Ser58 that regulates general endosomal binding and that the phosphorylation status of Ser631 mediates the endosomal shuttling between Rab5 and APPL1 subtypes. Taken together, these results reveal that MTMR2 compartmentalization and potential subsequent effects on endosome maturation and endosome signaling are dynamically regulated through MAPK-mediated differential phosphorylation events.

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues poses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.