Deregulation Of Hippo Pathway Research Articles

Identification of resistance mechanisms to small-molecule inhibition of TEAD-regulated transcription.

The Hippo tumor suppressor pathway controls transcription by regulating nuclear abundance of YAP and TAZ, which activate transcription with the TEAD1-TEAD4 DNA-binding proteins. Recently, several small-molecule inhibitors of YAP and TEADs have been reported, with some entering clinical trials for different cancers with Hippo pathway deregulation, most notably, mesothelioma. Using genome-wide CRISPR/Cas9 screens we reveal that mutations in genes from the Hippo, MAPK, and JAK-STAT signaling pathways all modulate the response of mesothelioma cell lines to TEAD palmitoylation inhibitors. By exploring gene expression programs of mutant cells, we find that MAPK pathway hyperactivation confers resistance to TEAD inhibition by reinstating expression of a subset of YAP/TAZ target genes. Consistent with this, combined inhibition of TEAD and the MAPK kinase MEK, synergistically blocks proliferation of multiple mesothelioma and lung cancer cell lines and more potently reduces the growth of patient-derived lung cancer xenografts in vivo. Collectively, we reveal mechanisms by which cells can overcome small-molecule inhibition of TEAD palmitoylation and potential strategies to enhance the anti-tumor activity of emerging Hippo pathway targeted therapies.

Genetic Alterations and Deregulation of Hippo Pathway as a Pathogenetic Mechanism in Bone and Soft Tissue Sarcoma.

The Hippo pathway is an evolutionarily conserved modulator of developmental biology with a key role in tissue and organ size regulation under homeostatic conditions. Like other signaling pathways with a significant role in embryonic development, the deregulation of Hippo signaling contributes to oncogenesis. Central to the Hippo pathway is a conserved cascade of adaptor proteins and inhibitory kinases that converge and regulate the activity of the oncoproteins YAP and TAZ, the final transducers of the pathway. Elevated levels and aberrant activation of YAP and TAZ have been described in many cancers. Though most of the studies describe their pervasive activation in epithelial neoplasms, there is increasing evidence pointing out its relevance in mesenchymal malignancies as well. Interestingly, somatic or germline mutations in genes of the Hippo pathway are scarce compared to other signaling pathways that are frequently disrupted in cancer. However, in the case of sarcomas, several examples of genetic alteration of Hippo members, including gene fusions, have been described during the last few years. Here, we review the current knowledge of Hippo pathway implication in sarcoma, describing mechanistic hints recently reported in specific histological entities and how these alterations represent an opportunity for targeted therapy in this heterogeneous group of neoplasm.

Recurrent WWTR1 S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Wnt/β-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats.

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/β-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for β-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and β-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, β-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active β-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of β-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of β-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/β-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/β-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

Abstract IA17: Hippo pathway deregulation in cancers

Abstract The Hippo signaling pathway is an evolutionarily conserved pathway that plays a key role in the development and homeostasis of tissues and organs. The TEAD family of the transcription factors (TEAD1-4) are the major transcription factors, and YAP/TAZ are the transcription activators/ key effectors of the Hippo pathway. TEADs regulate many biologic processes, including development, tissue homeostasis, and tumorigenesis by regulating target genes involved in cellular proliferation and survival. Amplification or upregulation of YAP/TAZ/TEADs correlates with poor prognosis in cancer patients. While data over the past few years have demonstrated that the pathway is dysregulated in a spectrum of cancers, the underlying molecular mechanisms of regulation and localization are areas of active investigation. One such focus area that I will discuss during my talk is the regulation and localization of TEADs. TEADs contain a highly conserved nuclear localization signal (NLS) embedded in the DNA binding domain (DBD). Overexpression of an NLS mutant TEAD in a Hippo-dependent cancer cell line is dominant negative and suppresses cellular proliferation and tumor growth. Additionally, we have identified physiologic conditions where TEADs are expressed in the cytoplasm and the nucleus. By performing comparative immunoprecipitation-mass spectrometry between WT and NLS mutant TEAD, we found novel interacting partners for cytoplasmic and nuclear TEAD, thus uncovering novel roles for TEADs in cancer. Citation Format: Anwesha Dey. Hippo pathway deregulation in cancers [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA17.

Abstract PR08: Systematic pan-cancer analyses of Hippo pathway deregulation in cancer

Abstract Hippo signaling is a highly conserved, pleiotropic pathway that is recurrently deregulated across many cancers. With large-scale cancer genomics data, several efforts have been undertaken to quantify Hippo pathway activity in an unbiased manner. However, identifying a comprehensive catalog of Hippo pathway genes can be difficult due to the heterogenous nature of the disease and the limitations of any given study. Here we show the high concordance between different methods of quantifying Hippo pathway activity in cancers. Furthermore, we establish a first-principled approach by experimentally deriving a lineage-independent quantification by leveraging the transcriptomic profiles of nearly 900 cell lines from 28 tissue types. We applied these signature(s) to the genomic profiles of over 11,000 primary human cancer patients to identify several statistically significant biomarkers associated with Hippo pathway dysregulation, including NF2 mutations and deletions, SOX2 amplifications, and CDH1 mutations. To further assess therapeutic implications of pathway inhibition, we applied machine learning approaches to predict a tumor’s sensitivity to Hippo pathway inhibition based on its genomic profile, revealing that Hippo pathway inhibition may be a tractable approach in a wide spectrum of cancers. This abstract is also being presented as Poster B38. Citation Format: Matthew T. Chang, Thijs J. Hagenbeek, Jennifer Lacap, Zora Modrusan, Christiaan Klijn, Anwesha Dey. Systematic pan-cancer analyses of Hippo pathway deregulation in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR08.

Abstract IA12: Epigenetic deregulation of the Hippo pathway mediates NF-κB driven sarcomagenesis

Abstract Soft tissue sarcomas are an aggressive group of roughly 65 mesenchymal malignancies diagnosed in 200,000 people worldwide annually. Unlike in epithelial cancers, where novel targeted therapies have had a dramatic effect on patient survival, the treatment approach for sarcomas has not changed significantly in 25 years. Furthermore, recent sequencing efforts have revealed that consistent oncogenic mutations are rare in muscle cancers. Though an individual sarcoma tumor may express mutant CDK4 or PTEN, these mutations are not widely or uniformly found in sarcomas and are not unifying molecular factors associated with a given subtype. As a result, these tumors are not sensitive to most available targeted therapeutics, which specifically interfere with the functions of mutant oncogenic pathways. Therefore, we investigated alternate genetic/epigenetic mechanisms underlying sarcomagenesis to facilitate development of novel therapeutics. Our previous work showed that copy number loss of upstream regulators deactivates the Hippo pathway leading to stabilization of the Hippo effector, Yes-Associated Protein 1 (YAP1). YAP1 stabilization promotes tumorigenesis in multiple sarcoma subtypes including muscle-derived tumors like human undifferentiated pleomorphic sarcoma (UPS), which are the focus of our studies. Deletion of Yap1 in the LSL-KrasG12D/+; Trp53fl/fl (KP) autochthonous mouse model of UPS resulted in significantly decreased tumorigenesis. Though much work has been done to establish YAP1 as a major regulator of tumorigenesis, most of the related mechanistic studies have been done in epithelial cancer models. Thus, the functional role of the Hippo pathway in mesenchymal tumors is still being debated. We have now identified a critical mechanism of YAP1-mediated sarcomagenesis in UPS using ChIP-seq (H3K27Ac) and super-enhancer (SE) analysis. H3K27Ac ChIP-seq and super-enhancer analysis of human UPS tumors revealed that NF-κB, a major regulator of proliferation and differentiation in muscle progenitor myoblasts, is the most transcriptionally active pathway in UPS. Using gene set enrichment analysis (GSEA) of microarray gene expression studies comparing control (KP) and Yap1-deficient (KPY) murine UPS tumors, we determined that Yap1 modulates expression of many NF-κB pathway components. Additionally, we found that YAP1 is constitutively active in these tumors due to epigenetic silencing of its inhibitor Angiomotin (AMOT). Together, these data suggest that persistent YAP1-mediated NF-κB signaling promotes sarcomagenesis. In addition to Hippo pathway deregulation, several studies have recently shown that alterations in epigenetic status can promote sarcomagenesis. Certain pediatric sarcomas are linked to chromosomal translocations of transcription factor loci encoding chromatin remodeling factors. Copy number loss and deletion of chromatin modulators have also been found in some subtypes. These proteins influence chromatin structure and coordinate regulation of normal developmental transcriptional pathways, indicating that disruption of chromatin architecture may be a common event in sarcomagenesis. Our recent work showed that treatment with the epigenetic modulating drug Vorinostat (SAHA), a pan-HDAC inhibitor, leads to reexpression of HIF2α and a 50% reduction in UPS sarcomagenesis in vivo. These studies support the hypothesis that epigenetic modulation can reexert control over malfunctioning pathways and return key transcription factors like YAP1 and HIF2α to the expression and activity levels found in quiescent cells. Treatment with the epigenetic modulators (SAHA and JQ1) inhibited YAP1 transcription and restored expression of AMOT, resulting in near ablation of YAP1 expression. Interestingly, SAHA/JQ1 treatment initiated a muscle differentiation program and reduced tumorigenesis in vivo. Unexpectedly, we also found that YAP1 promotes NF-κB activity by suppressing transcriptional oscillation of USP31, a newly identified upstream regulator of NF-κB. Both YAP1 shRNA and SAHA/JQ1 treatment induce USP31 mRNA oscillation over multiple days of treatment. Virtually nothing is known about USP31 beyond its structure and ability to inhibit NF-κB signaling. These findings suggest that Usp31, as well as other NF-κB components, might oscillate in quiescent or differentiating cells. Furthermore, we hypothesize that this oscillation plays a role in normal myoblast differentiation and is deregulated in YAP1-driven muscle tumors. Such oscillations are generally linked with the peripheral circadian clock, and disruptions in the clock are known to promote tumorigenesis. Furthermore, the link between an oscillating transcriptional program, circadian clock function, and normal muscle development has been established. However, our work is the first to suggest that perturbations in oscillating NF-κB targets and regulators are linked to sarcomagenesis. These data indicate that inappropriate expression of YAP1 may interfere with a temporally constricted NF-κB transcriptional program. This work establishes NF-κB, a key regulator of normal muscle development, as a pathway that becomes misregulated during sarcomagenesis through the aberrant activity of YAP1. Collectively, our data implicate multiple targets of YAP1 that could serve as useful biomarkers in UPS and provide the mechanistic rationale for epigenetic therapy for the treatment of this disease. Citation Format: T. S. Karin Eisinger. Epigenetic deregulation of the Hippo pathway mediates NF-κB driven sarcomagenesis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA12.

Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR.

Abstract 365: Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma

Abstract Hippo/YAP1 signaling pathway is a tumor suppressive pathway that controls the organ size by modulating the cell growth, proliferation and apoptosis and is conserved from Drosophila to mammals. In mammals, the Hippo tumor suppressor pathway consists of cascade of kinases in which MST1/2 phosphorylates and activates LATS1/2. The latter phosphorylates the oncogenic transcriptional coactivators YAP1 and TAZ, leading to their cytoplasmic retention by 14-3-3 proteins and/or degradation. Inactivation of MST and LATS kinases allows YAP1 and/ or TAZ nuclear translocation and subsequent activation of their target genes. Deregulation of Hippo pathway can induce tumors in model organisms and occurs in wide range of human cancers including melanoma. Merlin, a key component of this pathway which inhibits YAP1, is mutated/deleted in 8% of melanoma. Majority of uveal melanomas are driven by Gq/11 mutations that trigger YAP1 nuclear translocation, promoting tumor growth. High levels of YAP1 in BRAFV600E mutant tumors confer resistance to RAF- and MEK- targeted therapy in patients. Our results show that YAP1 level is elevated in melanoma and the YAP1 inhibitor verteporfin alone or in combination with B-RAF inhibitor PLX4720 reduces the viability, invasion and anchorage-independent growth of B-RAF V600E mutant SK-MEL-28 and SK-MEL-5 cells. In addition, verteporfin treatment also reduced the viability of PLX4720 resistant 1205 cells. Western blot analysis of verteporfin and PLX4720 treated SK-MEL-28 and SK-MEL-5 cells displayed reduced levels of YAP1, B-RAF, pERK, MEK and pMEK. We also report a novel physical interaction between YAP1 and B-RAF; this could be detected using double immunofluorescence and immunoprecipitation-western blotting techniques in both B-RAF V600E mutant and N-Ras mutant melanoma cells. Proximity ligation assays on tissue microarray showed that YAP1-B-RAF interaction is elevated in metastatic melanoma compared to normal skin. These novel findings highlight the crosstalk between B-RAF and Hippo/YAP1 signaling which might have a potential role in melanoma development and progression. Further, experiments are in progress to elucidate the functional significance of YAP1-B-RAF interaction in melanoma. Citation Format: Mohan Kumar Durai Raj, Jonathan Nguyen, Namrata Bora-singhal, Jane Messina, Geoffrey Gibney, Srikumar Chellappan. Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2017-365

The SRF-YAP-IL6 axis promotes breast cancer stemness

The SRF-YAP-IL6 axis promotes breast cancer stemness

The LATS2 tumor suppressor inhibits SREBP and suppresses hepatic cholesterol accumulation.

The Hippo signaling pathway is a major regulator of organ size. In the liver, Hippo pathway deregulation promotes hyperplasia and hepatocellular carcinoma primarily through hyperactivation of its downstream effector, YAP. The LATS2 tumor suppressor is a core member of the Hippo pathway. A screen for LATS2-interacting proteins in liver-derived cells identified the transcription factor SREBP2, master regulator of cholesterol homeostasis. LATS2 down-regulation caused SREBP activation and accumulation of excessive cholesterol. Likewise, mice harboring liver-specific Lats2 conditional knockout (Lats2-CKO) displayed constitutive SREBP activation and overexpressed SREBP target genes and developed spontaneous fatty liver disease. Interestingly, the impact of LATS2 depletion on SREBP-mediated transcription was clearly distinct from that of YAP overexpression. When challenged with excess dietary cholesterol, Lats2-CKO mice manifested more severe liver damage than wild-type mice. Surprisingly, apoptosis, inflammation, and fibrosis were actually attenuated relative to wild-type mice, in association with impaired p53 activation. Subsequently, Lats2-CKO mice failed to recover effectively from cholesterol-induced damage upon return to a normal diet. Additionally, decreased LATS2 mRNA in association with increased SREBP target gene expression was observed in a subset of human nonalcoholic fatty liver disease cases. Together, these findings further highlight the tight links between tumor suppressors and metabolic homeostasis.

Emerging role of Hippo pathway in gastric and other gastrointestinal cancers.

More evidence has underscored the importance of Hippo signaling pathway in gastrointestinal tissue homeostasis, whereas its deregulation induces tumorigenesis. Yes-associated protein 1 (YAP1) and its close paralog TAZ, transcriptional co-activator with a PDZ-binding motif, function as key effectors negatively controlled by the Hippo pathway. YAP1/TAZ exerts oncogenic activities by transcriptional regulation via physical interaction with TEAD transcription factors. In various cancers, Hippo pathway cross-talks with pro- or anti-tumorigenic pathways such as GPCR, Wnt/β-catenin, Notch and TGF-β signaling and is deregulated by multiple factors including cell density/junction and microRNAs. As YAP1 expression is significantly associated with poor prognosis of gastric and other gastrointestinal cancers, detailed delineation of Hippo regulation in tumorigenesis provides novel insight for therapeutic intervention. In current review, we summarized the recent research progresses on the deregulation of Hippo pathway in the gastrointestinal tract including stomach and discuss the molecular consequences leading to tumorigenesis.

Germline polymorphisms in genes involved in the Hippo pathway as recurrence biomarkers in stages II/III colon cancer.

The Hippo pathway regulates tissue growth and cell fate. In colon cancer, Hippo pathway deregulation promotes cellular quiescence and resistance to 5-Fluorouracil. In this study 14 polymorphisms in 8 genes involved in the Hippo pathway (MST1, MST2, LATS1, LATS2, YAP, TAZ, FAT4 and RASSF1A) were evaluated as recurrence predictors in 194 patients with stages II/III colon cancer treated with 5-Fu-based adjuvant chemotherapy. Patients with a RASSF1A rs2236947 AA genotype had higher 3-year recurrence rate than patients with CA/CC genotypes (56% vs 33%, HR: 1.87; p=0.017). Patients with TAZ rs3811715 CT or TT genotypes had lower 3-year recurrence rate than patients with a CC genotype (28% vs 40%; HR: 0.66; p=0.07). In left-sided tumors, this association was stronger (HR: 0.29; p=0.011) and a similar trend was found in an independent Japanese cohort. These promising results reveal polymorphisms in the Hippo pathway as biomarkers for stage II and III colon cancer.

Germline polymorphisms in genes involved in the Hippo pathway to predict recurrence in locally advanced colon cancer.

e14505 Background: The Salvador-Warts-Hippo pathway regulates tissue growth and cell fate. The Hippo pathway deregulation has been described in several cancer types and promotes stem cell maintenan...

Hippo Pathway (HP) Activity In Acute Myelogenous Leukemia (AML): Different Prognostic Implications Of TAZ versus YAP Inactivation By Phosphorylation

BackgroundThe highly evolutionarily conserved Hippo pathway (HP) regulates proliferation, apoptosis polarity and stem cell maintenance. In Humans, TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes associated protein) are the functional effectors, regulating gene expression by co-activating several transcription factors (RUNX, TEADS, SMADS). Phosphorylation inactivates both, causing expulsion from the nucleus and either proteosomal degradation or sequestration by 14-3-3 or membrane protein complexes. A 4 protein complex of LATS 1/2 kinase, MST1/2, MOB and SAV1, phosphorylates YAP and TAZ, and is regulated by many pathways including Neurofibromin 2 (NF2) , and WNT, and by cell-cell contact. YAP and TAZ share many binding and interaction partners, sharing overlapping functions, but they also have exclusive interactions leading to some exclusive functions, notably in SMAD family and TGFβ regulation. Deregulation of HP is associated with numerous solid tumors (breast, lung, colorectal, ovary, liver), with upregulation conferring an adverse prognosis, but mutations of HP components is rare, suggesting other mechanism for deregulation. The role of the HP pathway in AML is undefined and similar to most solid tumors, mutations in AML were not observed by TCGA. MethodsTo define the role of the HP in AML we used reverse phase protein array (RPPA) technology to print protein from leukemia enriched cells from 511 newly diagnosed AML patients. The RPPA was probed with 231 antibodies, including antibodies against both total and phosphorylated HP components NF2, YAP (p-Serine 127) and TAZ (p-Serine 89). We could not validate antibodies for LATS 1 or 2. ResultsLevels of total NP2, TAZ YAP were often above that of normal CD34+ cells in 30%, 15% and 17% of cases. Levels of phosphorylated NF2 (p=0.07, 23%) and YAP (p=0.009, 10%) were often above, and those of pTAZ (p= 0.008, 29%) below that of normal CD34+ cells. Higher levels of pTAZ were associated with FAB MO and M1 subtypes, higher CD7 and CD34 expression and higher % marrow blasts. Higher levels of pYAP were associated with lower % marrow and peripheral blood blasts and with cases transformed from RAEBT and CMML. The optimum number of principal components for these 6 antibodies was two, depending on the phosphorylation state of YAP and TAZ. Another statistical method determined the optimal number of patient groups to be 6 (figure 1) with expression 1) Pan Off (19% n=79, Yellow in Fig.) 2) High Total (2%, n =8, Dark Pink.) with very high levels of NF2, TAZ and YAP and high pNF2, 3) Active HP (30% n=123, Blue), with moderate total levels but scant phosphorylation, 4) YAP inactive (19%, n=78, Light Pink) and 5) TAZ inactive (22.5%, n=93, Purple) where only one is phosphorylated and 6) Both inactivated (9% n=37, Green) with both heavily phosphorylated. The TAZ inactive motif was prognostically adverse for overall survival (OS) (Median 30 weeks p=0.009) compared to active, pYAP, both inactivated (median 48-60 weeks), while the High Total pattern had longer OS (median 224 wks). Notably, high pTAZ alone or with pYAP was associated with inferior response and OS in patients treated with demethylating and histone deacetylating drugs. Favorable cytogenetics patients with active HP, High pTAZ or both inactive, treated with fludarabine-HDAC had shorter OS and significantly shorter remission duration (52-70 weeks vs. not reached). High pTAZ was associated with higher SMAD family levels and with unfavorable cytogenetics as well as with higher levels of phosphorylated (inactivated) β-Catenin, likely due to cytoplasmic localization. [Display omitted] ConclusionsHP proteins are heterogeneously expressed in AML and key effectors YAP and TAZ are differentially phosphorylated. Differential phosphorylation of YAP and TAZ has not been previously reported. This suggests that active YAP in the presence of inactive TAZ is inducing expression of genes that provide proliferative or survival advantages to leukemic blasts. The observation that the Highly active HP group did very well may be identifying a high proliferative group that is more susceptible to cycle specific chemotherapy, similar to Burkitt's leukemia. In summary, this data suggest that the HP pathway is active in AML and has a previously undescribed pattern of separate phosphorylation of YAP and TAZ. Disclosures:No relevant conflicts of interest to declare.