PTEN Deletion Research Articles

Abstract 5295: Combined loss of tristetraprolin (TTP) and PTEN leads to aggressive prostate cancer and hypersensitivity to nuclear factor kappa B (NFkB) inhibition

Abstract We have reported that loss of tristetraprolin (TTP, gene name ZFP36), an RNA binding protein which modulates mRNA stability increased NFκB activation and is associated with higher rates of disease recurrence in prostatectomy series. Given TTP loss occurs in early prostate cancer we sought to examine the clinical and biological impact of TTP loss combined with PTEN loss, which also occurs in early prostate cancer. IHC revealed an increased risk of relapse with loss of one gene alone and a significantly higher risk of relapse with loss of both TTP and PTEN, (HR 4.50, p=0.04 on MVA adjusting for standard clinicopathologic factors). These findings were confirmed by analysis of RNA profiles of two publicly available prostatectomy series. Engineering ZFP36 deletion specific to prostate luminal cells in vivo induced high-grade prostatic intraepithelial neoplasia (HG-PIN), and when combined with PTEN deletion (P:Z) resulted in rapid progression to invasive adenocarcinoma associated with increased proliferation and development of micrometastases. Combined loss of PTEN and ZFP36 in mice significantly reduced overall survival (53.4 (P:Z+/-) and 49.6 (P:Z-/-) weeks versus 66.9 weeks in PTEN-/- (p<0.0001). Further, surgical castration of mice demonstrated that combined loss of ZFP36 and PTEN significantly accelerated progression to castration resistance versus PTEN alone (median survival, 56.1 (P:Z+/-) and 53.9 (P:Z-/-) weeks versus all PTEN-/- mice still alive by 70 weeks). Ex vivo analysis of organoid growth of the tumors revealed presence of budding with PTEN-/- with ZFP36-/- and almost none with PTEN-/-. These findings were recapitulated with GEMM-derived allograft tumor growth with tumor sizes 20 weeks after castration being 200mm3 in PTEN-/- versus 900mm3 with [PTEN-/- + TTP-/-] versus 600mm3 for PTEN-/- and 1200mm3 for [PTEN-/- + TTP-/-] after sham castration (all comparisons p < 0.05). When the PTEN/ZFP36 null allograft was treated with the NFκB inhibitor dimethylaminoparthenolide (DMAPT, D), tumor sizes 36 days after treatment were: vehicle - 2000mm3, castration (C) - 1500mm3; D alone - 600mm3; C + D - 500mm3 (all comparisons p<0.05; except D vs C + D; p=0.07). These findings were supported by cell death assays of organoid models for the GEMMs. The hypersensitivity of PTEN/ZFP36 null cell line to DMAPT was seen with dose response curves with DMAPT LD50 being 4.52 μM PTEN-/-; 0.76 μM [PTEN -/- + TTP-/-] and 8.66 μM for [PTEN-/- + TTP-/- + silenced p65]. In conclusion, combined loss of PTEN and TTP results in prostate cancer that is clinically more aggressive than PTEN loss alone and this was corroborated by GEMM models. Namely, PTEN/TTP null tumors were more aggressive, minimally responsive to castration, demonstrated activation of NFκB and hypersensitivity to DMAPT. This work supports the planned clinical trials of DMAPT in castration resistant prostate cancer Citation Format: Christopher J. Sweeney, Katherine Morel, Anis Hamid, Leigh Ellis. Combined loss of tristetraprolin (TTP) and PTEN leads to aggressive prostate cancer and hypersensitivity to nuclear factor kappa B (NFkB) inhibition [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 5295.

Abstract 928: USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy and one of the most common causes of cancer mortality among women worldwide. Ubiquitin-Specific Peptidase 13 (USP13) gene copy is strongly amplified in human epithelial ovarian cancer, and high USP13 expression is correlated with poor survival outcomes, yet its pathological contribution to ovarian tumorigenesis remains unknown. Here, we crossed a conditional Usp13 overexpressing knock-in mouse with a conditional knockout of Trp53 and Pten mouse, and generated a novel ovarian cancer genetically engineered mouse model (GEMM), which closely recapitulates the genetic changes driving ovarian cancer in humans. Overexpression of USP13 with deletion of Trp53 and Pten in murine ovarian surface epithelium accelerated ovarian tumorigenesis and led to decreased survival in mice. Notably, USP13 greatly enhanced peritoneal metastasis of ovarian tumors with frequent development of hemorrhagic ascites. The primary and metastatic tumors exhibited morphology and clinical behavior similar to human high-grade serous ovarian cancer. Co-inhibition of USP13 and AKT significantly decreased the viability of the primary murine ovarian cancer cells isolated from the GEMM. USP13 also increased the tumorigenic and metastatic abilities of primary murine ovarian cancer cells in a syngeneic mouse study. These findings suggest a critical role of USP13 in ovarian cancer development and reveal USP13 as a potential therapeutic target for ovarian cancer. Citation Format: Juntae Kwon, Hyungja Choi, Anna D. Ware, Bernadette Cecilia Morillo, Haiyang Wang, Cecil Han. USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model [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 928.

Abstract 1181: PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy

Abstract Background: The clinical development of farnesyltransferase inhibitors (FTIs) as targeted therapy for HRAS-mutant cancers has demonstrated mixed responses dependent on cancer type. Co-occurring mutations may affect tumor response, supported by previous studies demonstrating that NF1 mutations confer resistance to HRAS inhibition by the FTI tipifarnib in thyroid cancer mouse models. We aimed to determine if PI3K pathway activating mutations altered responses to targeted therapy in HRAS-mutant cancers. Methods: Targeted sequencing data from MSK-IMPACT cohort and DFCI-GENIE (Version 9.0) database was used to investigate co-mutations amongst HRAS-mutant cancers. Fisher’s exact test was used to determine co-altered mutations found predominantly in HRAS-mutant cancers relative to respective KRAS- and NRAS-mutant cancers. ‘RASless’ (KRASlox/HRASKO/NRASKO) mouse embryonic fibroblasts (MEFs) were obtained that in the presence of 600nM tamoxifen (4OHT) resulted in a KRAS knock-out. ‘Rasless’ MEFs were transfected with HRASG13R to create a system for testing sensitivity to FTIs in the presence or absence of WT KRAS, or with concurrent PTEN loss generated by CRISPR-Cas9 technology. Results: A greater proportion of HRAS-mutant cancers had co-altered mutations (48.8%) in genes encoding effectors in the MAPK, PI3K or RTK pathways compared to KRAS- and NRAS-mutant cancers (41.4% and 38.4%, respectively; p<0.05). PTEN mutations were more prevalent in HRAS-mutant NSCLC (21%) compared to KRAS- and NRAS-mutant NSCLC (1% and 2%, respectively; p<0.05). Non-transfected MEFs were sensitized to tipifarnib by introduction of a HRASG13R allele in non-4OHT (IC50: MEF= >3uM, HRASG13R = 324.7nM) and 4OHT (IC50: MEF= >3uM, HRASG13R= 0.62nM; p<0.001) conditions, indicating that WT KRAS confers a relative resistance to the inhibitory effects of the FTI on HRAS. PTEN loss-of-function mutations led to tipifarnib resistance in HRASG13R MEFs in the absence (IC50: >3uM; p<0.001) or presence of 4OHT (IC50: 213.6nM; p<0.001). Combined treatment of HRASG13R/PTEN MEFs with the PIK3CB-specific inhibitor AZD8186 and tipifarnib sensitized cells in non-4OHT (IC50- 100nM:100nM Tipifarnib:AZD8186) and 4OHT (IC50- 100nM:10nM Tipifarnib:AZD8186) conditions. Conclusions: Co-altered mutations of MAPK, PI3K or RTK effectors are found more commonly in HRAS than in KRAS or NRAS-mutant cancers. Co-alteration of PTEN preferentially associated with HRAS-mutations in NSCLC. Deletion of PTEN resulted in resistance to FTI targeted therapy in vitro. Co-altered mutations may predict sensitivity and resistance to FTIs and guide clinical trial design. Citation Format: Aradhya Nigam, Walid K. Chatila, Gnana P. Krishnamoorthy, Alan L. Ho, James A. Fagin, Nikolaus D. Schultz, Brian R. Untch. PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy [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 1181.

Abstract 3816: Functional atlas of prostate mesenchyme

Abstract Prostate cancer has a heterogeneous prognosis, and genetic alterations alone do not fully explain clinical behavior. We previously characterized the stroma of localized human prostates by Laser Capture Microdissection, and found that stroma was substantially different in prostates with and without tumor. Furthermore, a stromal gene signature reflecting bone remodeling was upregulated in high compared to low Gleason grade cases. To determine how stromal cells contribute to carcinogenesis and progression we study whether specific genetic alterations in the epithelium induce unique stromal changes. To do this, we utilized Genetically Engineered Mouse Models (GEMMs) representing common prostate cancer mutations and compared these to their wild-type conterparts: the Tmprss2-ERG fusion knock-in murine model induces histological alterations in the stroma in the absence of an epithelial phenotype; the Pten deletion mouse model (PtenKO) results in prostate intraepithelial neoplasia (PIN) but not invasive cancer; the Hi-Myc GEMM, leads to PIN and subsequently invasion; and the Pb4-Cre +/-;Pten f/f; LSL-MYCN +/+; Rb1 f/f (MNRPDKO) mouse model that leads to neuroendocrine prostate cancer (NEPC). We generated a comprehensive single-cell transcriptomic atlas of the mouse prostate cancer mesenchyme in these models. Using deep generative modeling followed by graph-based clustering and gene regulatory network inference, six (6) distinct subsets of fibroblasts and two (2) subsets of smooth muscle cells (myofibroblasts and pericytes) were identified. Notably, some subsets were common across all GEMMs and WT mice, while others aligned with specific genotypes. Moreover, we found a variable pattern of positive and negative Ar expressing cells between genotypes. Analysis by CellphoneDB of mesenchymal-epithelial communications revealed the complex cross-talk between mutated epithelial cells and the tumor microenvironment. Multiplex immunofluorescence phenotyping of mesenchymal cell confirmed the cluster subtypes by both expression and spatial location. Finally, stromal transcripts defining mesenchymal cluster subtypes associated with Tmprss2-ERG were conserved between mouse and human genotypes.This study lays the groundwork for understanding and ultimately targeting stromal-epithelial interactions in prostate cancer. Citation Format: Hubert Pakula, Ryan Carelli, Nicolo Fanelli, Madhavi Jere, Caitlin Unkenholz, Mohamed Omar, Caroline Ribeiro- Fidalgo, Filippo Pederzoli, Cory Abate-Shen, David S. Rickman, Brian Robinson, Luigi Marchionni, Massimo Loda. Functional atlas of prostate mesenchyme [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 3816.

Abstract 1667: Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with no improvements in treatment options for RMS patients over the past four decades. Therefore, it is critical to understand the fundamental processes underlying RMS tumorigenesis. RMS is divided into two major histologic subtypes - alveolar and embryonal RMS. Nearly all alveolar RMS express oncogenic fusions of PAX3-FOXO1 or PAX7-FOXO1 whereas embryonal RMS are not driven by these fusion proteins. Instead, embryonal or fusion-negative (FN-RMS) are molecularly heterogeneous. Approximately one-third of fusion-negative RMS (FN-RMS) patients have copy number loss of PTEN (phosphatase and tensin homolog), and approximately 90% of tumors are hypermethylated at the PTEN promoter leading to decreased PTEN expression. This indicates a near universal role for PTEN loss in FN-RMS, but the functional role of PTEN is still unclear. To determine PTEN’s function in FN-RMS, we bred Ptenflox alleles into our aP2-Cre;SmoM2 (ASPWT) FN-RMS mouse model to obtain aP2-Cre;SmoM2;Ptenflox/flox mice (ASPcKO). Conditional Pten deletion accelerated tumorigenesis and produced a tumor with a less differentiated histological appearance, much like human FN-RMS. Interestingly, in PtenWT tumors, we found predominant PTEN immunoreactivity within the nucleus suggesting a role for nuclear PTEN in FN-RMS. Transcriptome analyses revealed robust gene expression changes between the ASPWT and ASPcKO tumors. The top overexpressed gene in ASPcKO tumors was Dbx1 (Developing brain homeobox 1), a homeobox transcription factor with no known cancer function but involved in innate behavioral processes such as breathing. We found FN-RMS patient-derived xenografts are dependent on DBX1 expression, and that DBX1 expression is controlled by PAX7 (Paired Box 7). PAX7 is a transcription factor expressed in satellite cells and maintains a de-differentiated state in FN-RMS. PAX7 expression is also increased in our ASPcKO tumors, and we show that human FN-RMS cells are dependent on PAX7 expression for proliferation. This suggests PTEN loss in FN-RMS engages a new transcriptional program necessary for FN-RMS survival. To determine if Pax7 loss can rescue the deleterious effects of Pten loss in our murine FN-RMS model, we deleted both Pten and Pax7 in our aP2-Cre;SmoM2 mice (ASPcKOP7cKO). ASPcKOP7cKO tumor onset kinetics resembled tumors with wild-type PTEN and were negative for skeletal muscle markers MYOD1 and MYOGENIN. However, these tumors were positive for leiomyosarcoma markers smooth muscle actin and CALDESMON. Together, our data suggests PTEN and PAX7 have a synthetic essential relationship in FN-RMS and that PAX7 is a proliferative driver and lineage dependency for FN-RMS tumors. This work also illustrates the power of murine models to unravel the genetic dependencies underlying both tumor maintenance and identity. Citation Format: Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Myron K. Evans, Kristin B. Reed, Madeline Bush, Jason A. Hanna, Catherine J. Drummond, Matthew C. Maguire, Patrick J. Leavey, David Finkelstein, Hongjian Jin, Patrick A. Schreiner, Jerold E. Rehg, Mark E. Hatley. Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity [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 1667.

Multicentric Glioma: An Ideal Model to Reveal the Mechanism of Glioma.

As a special type of glioma, multicentric glioma provides an ideal pathological model for glioma research. According to the stem-cell-origin theory, multiple lesions of multicentric glioma share the same neuro-oncological origin, both in gene level and in cell level. Although the number of studies focusing on genetic evolution in gliomas with the model of multicentric gliomas were limited, some mutations, including IDH1 mutations, TERTp mutations and PTEN deletions, are found to be at an early stage in the process of genetic aberrance during glioma evolution based on the results of these studies. This article reviews the clinical reports and genetic studies of multicentric glioma, and intends to explain the various clinical phenomena of multicentric glioma from the perspective of genetic aberrance accumulation and tumor cell evolution. The malignant degree of a glioma is determined by both the tumorigenicity of early mutant genes, and the stemness of early suffered cells.

Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN-deficient endometrial carcinoma

Loss of phosphatase and tensin homolog (PTEN) impairs DNA double-strand repair and confers sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPis). However, PARPis also hyperactivate the MAPK and PI3K/AKT/mTOR pathways in PTEN-deficient endometrial carcinoma (EC), which allows the emergence of PARPi resistance. BCL-2–associated death promoter (BAD), integrates the common cell survival effects of the RAS/MEK/MAPK and PI3K/AKT/mTOR pathways. Herein, it was observed that increased BADSer99 (BADS99) phosphorylation in EC cells was significantly associated with PTEN-deficient status. Forced expression of phosphorylation deficient human BADS99A in PTEN-deficient EC cells significantly increased CASPASE 3/7 activity and decreased EC cell viability. Using NPB as a pharmacological inhibitor of pBADS99 phosphorylation, it was demonstrated that NPB synergized with PARPis (Olaparib, Rucaparib and Talazoparib) to enhance PARPi IC50 up to 60-fold and decreased survival, foci formation, and growth in 3D ex vivo culture of PTEN-deficient EC cells. Combined NPB-PARPi treatment of PTEN-deficient EC cells stimulated apoptosis and promoted DNA damage by impairment of homologous recombination. Using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease system it was demonstrated that deletion of PTEN in PTEN replete EC cells enhanced the efficacy of combined NPB-PARPi treatment. Furthermore, combined inhibition of BADS99 phosphorylation and PARP ablated xenograft growth of PTEN-deficient EC cells. Similarly, a combination of NPB and PARPis significantly suppressed the growth of PTEN deficient patient-derived EC organoids. Hence, combined inhibition of BADS99 phosphorylation and PARP represents a rational and efficacious strategy to improve the prognosis of recurrent EC patients.

Translational modeling for patients with RAS mutant tumors: Profiling the dual-MEK inhibitor IMM-1-104 in a humanized 3D assay.

e15084 Background: Elevated RAS-RAF-MEK-ERK (MAPK pathway) signaling is observed in over half of all solid human tumors, and mutations in RAS or RAF account for a large fraction. Given MEK’s unique position in the MAPK cascade, it remains an attractive target in cancer. However, FDA-registered MEK inhibitors are susceptible to pathway reactivation events that limit their use to RAF mutant disease and cause on-target toxicities stemming from chronic target engagement. IMM-1-104 is a novel, allosteric dual-MEK inhibitor designed for better applicability to RAS mutant tumors by preventing MEK reactivation. Endowed with a short plasma half-life, IMM-1-104 promotes deep cyclic inhibition with a near-zero drug trough, affording normal cells a chance to recover between doses. Methods: We characterized IMM-1-104’s pharmacologic activity across 52 tumor cell lines that spanned 11 distinct tumor types in a humanized, ECM-based 3D tumor growth assay (3D-TGA). The 3D-TGA has better predicted in vivo tumor responses versus 2D culture and more accurately reflects human tumor biology. Tumor models were categorized based on in vivo drug PK limits as sensitive to IMM-1-104 (EC50 < 1uM), intermediate (1uM≤EC50≤10uM and ≥25% inhibition at 10uM) or resistant otherwise. Models were evaluated by whole exome sequencing, along with RNA sequencing in the 3D context, to profile determinants of sensitivity and resistance and to prioritize patient populations most likely to respond to IMM-1-104. Results: Models sensitive to IMM-1-104 were enriched for MAPK driver mutations, consistent with pathway addiction. We reasoned that activation of parallel compensatory pathways that can reduce reliance on MAPK signaling may increase the likelihood of resistance to IMM-1-104. Pathways and genes suspected of contributing to resistance helped refine signatures based on 3D-TGA outcome data. Models with a MAPK driver mutation and compensatory mutations such as PIK3CA or PTEN deletion were more likely to show intermediate response than those with a greater addiction to MAPK drivers. Models lacking a clear MAPK driver mutation but harboring other putative resistance alterations were more likely to be resistant in the 3D-TGA. Conclusions: To better understand the relevance of tumor model responses in the 3D-TGA relative to RAS mutant patient populations, we computationally compared tumor model data to patient somatic alterations, identified in the public resource GENIE, which has cataloged the molecular profiles of over 100,000 cancer patients. Based on model-to-patient molecular mapping, we identified biomarker-defined subsets of sensitive KRAS mutant lung and colorectal models. The most broadly sensitive patient-aligned models in the 3D assay were KRAS mutant pancreatic cancer and NRAS mutant melanoma patients, supporting the inclusion of such patients in planned clinical studies of IMM-1-104.

Genomic landscape and peripheral blood biomarkers of advanced triple-negative breast cancer treated with immune checkpoint blockade: An exploratory analysis of the TQB2450-Ib-07 trial.

1080 Background: Immune checkpoint inhibitor (ICI) has emerged as a novel therapeutic option for advanced triple-negative breast cancer (aTNBC). However, no robust biomarker indicative of clinical outcomes has been identified. Herein, we portraited the genomic landscape and explored the biomarkers for patients with aTNBC receiving ICI-based therapy. Methods: This is a prospective, multicenter, phase 1b clinical trial (NCT03855358) to assess the efficacy and safety profiles of TQB2450, a humanized monoclonal PD-L1 antibody, plus anlotinib in pretreated TNBC. Eligible patients undergo liquid biopsy at baseline and the timepoint of disease progression. NGS-based assay was performed based on circulating tumor DNA (ctDNA) in the bloodstream. Meanwhile, results of laboratory blood tests were dynamically collected and blood markers, including neutrophil to lymphocyte ratio (NLR), lymphocyte to monocyte ratio (LMR), and platelet to lymphocyte ratio (PLR), were successively evaluated. The primary endpoints were progression-free survival (PFS) and clinical efficacy assigned via guidelines for response criteria for use in trials testing immunotherapies (iRECIST). Results: Between May 29, 2019, and December 31, 2020, 34 patients with aTNBC were enrolled. Gene alternations primarily comprised mutation, amplification, or deletion of TP53, MLL3, DNMT3A, PI3KCA, EP300, PTEN, LRP1B, MDM2, and NCOR1. The median maximum somatic allele frequency (MSAF) was 9.97% significantly indicative of PFS, which was 3.58 months for the MSAF-high group and 13.34 months for the MSAF-low group (P = 3e-04), respectively. Else, a strong association was also signified between MSAF and tumor shrinkage (CR/PR vs. SD/PD, P = 0.012). For blood tumor mutation burden (bTMB), the median was 6.72 muts/Mb, which the bTMB-low group was suggestive of a better PFS (11.09 months vs. 5.52 months, P = 0.007), yet no obvious association existing in terms of clinical response. Dynamic analysis revealed that a decline in MSAF was significantly associated with a better PFS (7.10 months vs. 2.74 months, P = 0.018), while no correlations were detected between bTMB and PFS. Based on NLR week 2/0 of 0.95, PFS was significantly worse in the NLR-low group (11.0 months vs. 3.5 months, P = 0.006) and likely distinguished the clinical response (CR/PR vs. SD vs. PD, P = 0.049; non-PD vs. PD, P = 0.022). Moreover, NLR week 2/0 could notably foretell the clinical response for patients with aTNBC with the AUC of 0.82 (0.61-1.00). No comparable utilities were identified regarding LMR and PLR. Conclusions: For aTNBC treated with ICI, MSAF tended to be a robust indicator for both PFS and clinical response. NLR week 2/0 presented a favorable profile indicative of PFS as well as a strong predictor for clinical responsiveness of patients with aTNBC receiving immune checkpoint blockade. Clinical trial information: NCT03855358.

Genomic correlates of acquired resistance to PD-(L)1 blockade in patients with advanced non-small cell lung cancer (NSCLC).

9021 Background: Despite improvements in survival with immune checkpoint inhibition (ICI), the majority of patients develop acquired resistance to ICI after an initial benefit. However, the mechanisms underlying acquired resistance to ICI in NSCLC are largely unknown. Methods: Patients with advanced NSCLC treated with ICI at the Dana-Farber Cancer Institute (DFCI), and whose tumors underwent genomic profiling before and after ICI, with no intervening therapies, were included. Mutations, tumor mutational burden (TMB), copy number variations (CNVs), and PD-L1 tumor proportion score (TPS) were compared between pre- and post-ICI samples. Acquired resistance was defined as the development of disease progression after an initial objective response, or stable disease ≥3 months with PD-(L)1 blockade. Results: Among 1763 patients with advanced NSCLC who received ICI, 45 had matched pre- and post-ICI tissue samples available for genomic profiling. Putative mechanisms of resistance were identified in 55% of cases (N = 25). Five patients (20%) acquired an STK11 mutation, one patient (4%) acquired a KEAP1 mutation, and another patient (4%) developed concurrent KEAP1 and SMARCA4 mutations. A patient (4%) with KRAS G12C-mutant NSCLC developed concurrent STK11 and KEAP1 mutations at resistance. In 3 cases (12%) with pre-existing STK11 or KEAP1 mutations prior to ICI administration, we identified acquired copy losses of STK11 and KEAP1, respectively, resulting in bi-allelic inactivation of these genes. Acquired beta-2-microglobulin ( B2M) mutations were detected in 3 patients (12%), one of whom developed concurrent B2M copy loss, indicating bi-allelic inactivation. Eight additional patients (32%) developed B2M gene deletions. Other acquired alterations that have been implicated in ICI resistance included CDKN2A/B loss (N = 10, 40%), including 5 with bi-allelic deletion, acquired PTEN deletions (N = 5, 20%), and MDM2 amplification (N = 2, 8%). When we examined alterations in immune checkpoint genes, we identified acquired CD274 (PD-L1) and PDCD1LG2 (PD-L2) loss in 8% of cases (N = 2), and bi-allelic deletion in one case (4%). Intervening ICI did not affect TMB (median TMB: 8.7 [pre-ICI] vs 9.1 [post-ICI] mut/Mb, P = 0.6), PD-L1 expression (median PD-L1 TPS: 3% [pre-ICI] vs 5.0% [post-ICI] mut/Mb, P = 0.5), or aneuploidy levels (as fraction of genome altered [FGA]) (median FGA: 18.4% [pre-ICI] vs 21.1% [post-ICI], P = 0.2), indicating that acquired gene level CNVs were not a reflection of increased cancer aneuploidy. In a control cohort of 30 patients with pre- and post-chemotherapy matched samples which underwent genomic profiling, no acquired mutations in STK11, KEAP1, SMARCA4, or B2M were detected. Conclusions: Mechanisms of acquired resistance to PD-(L)1 blockade are heterogenous, and new therapeutic strategies are required to delay and overcome ICI resistance in patients with NSCLC.

The phosphatase PTEN links platelets with immune regulatory functions of mouse T follicular helper cells

Beyond a function in hemostasis and thrombosis, platelets can regulate innate and adaptive immune responses. Hyperactive platelets are frequently associated with multiple human autoimmune diseases, yet their pathogenic functions in these diseases have not been fully established. Emerging studies show an essential function of the phosphatase and tensin homolog (PTEN) in maintenance of immune homeostasis. Here, we show that mice with platelet-specific deletion of Pten, develop age-related lymphoproliferative diseases and humoral autoimmunity not seen in wildtype animals. Platelet-specific Pten-deficient mice have aberrant T cell activation, excessive T follicular helper (Tfh) cell responses and accumulation of platelet aggregates in lymph nodes. Transferred Pten-deficient platelets are able to infiltrate into the peripheral lymphoid tissues and form more aggregates. Moreover, Pten-deficient platelets are hyperactive and overproduce multiple Tfh-promoting cytokines via activation of the PDK1/mTORC2-AKT-SNAP23 pathway. Pten-deficient platelets show enhanced interaction with CD4+ T cells and promote conversion of CD4+ T cells into Tfh cells. Our results implicate PTEN in platelet-mediated immune homeostasis, and provide evidence that hyperactive platelets function as an important mediator in autoimmune diseases using mouse models.

Enhancers regulate 3′ end processing activity to control expression of alternative 3′UTR isoforms

Multi-UTR genes are widely transcribed and express their alternative 3′UTR isoforms in a cell type-specific manner. As transcriptional enhancers regulate mRNA expression, we investigated if they also regulate 3′UTR isoform expression. Endogenous enhancer deletion of the multi-UTR gene PTEN did not impair transcript production but prevented 3′UTR isoform switching which was recapitulated by silencing of an enhancer-bound transcription factor. In reporter assays, enhancers increase transcript production when paired with single-UTR gene promoters. However, when combined with multi-UTR gene promoters, they change 3′UTR isoform expression by increasing 3′ end processing activity of polyadenylation sites. Processing activity of polyadenylation sites is affected by transcription factors, including NF-κB and MYC, transcription elongation factors, chromatin remodelers, and histone acetyltransferases. As endogenous cell type-specific enhancers are associated with genes that increase their short 3′UTRs in a cell type-specific manner, our data suggest that transcriptional enhancers integrate cellular signals to regulate cell type-and condition-specific 3′UTR isoform expression.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Co-targeting B-RAF and PTEN Enables Sensory Axons to Regenerate Across and Beyond the Spinal Cord Injury.

Primary sensory axons in adult mammals fail to regenerate after spinal cord injury (SCI), in part due to insufficient intrinsic growth potential. Robustly boosting their growth potential continues to be a challenge. Previously, we showed that constitutive activation of B-RAF (rapidly accelerated fibrosarcoma kinase) markedly promotes axon regeneration after dorsal root and optic nerve injuries. The regrowth is further augmented by supplemental deletion of PTEN (phosphatase and tensin homolog). Here, we examined whether concurrent B-RAF activation and PTEN deletion promotes dorsal column axon regeneration after SCI. Remarkably, genetically targeting B-RAF and PTEN selectively in DRG neurons of adult mice enables many DC axons to enter, cross, and grow beyond the lesion site after SCI; some axons reach ∼2 mm rostral to the lesion by 3 weeks post-injury. Co-targeting B-RAF and PTEN promotes more robust DC regeneration than a pre-conditioning lesion, which additively enhances the regeneration triggered by B-RAF/PTEN. We also found that post-injury targeting of B-RAF and PTEN enhances DC axon regeneration. These results demonstrate that co-targeting B-RAF and PTEN effectively enhances the intrinsic growth potential of DC axons after SCI and therefore may help to develop a novel strategy to promote robust long-distance regeneration of primary sensory axons.

Loss of Arid1a and Pten in Pancreatic Ductal Cells Induces Intraductal Tubulopapillary Neoplasm via the YAP/TAZ Pathway

Background & AimsPancreatic ductal adenocarcinoma (PDAC) arises from several types of premalignant lesions, including intraductal tubulopapillary neoplasm (ITPN); however, the molecular pathogenesis of ITPN remains unknown. MethodsWe performed studies with Hnf1b-CreERT2; Ptenf/f; Arid1af/f mice to investigate the consequence of genetic deletion of Arid1a in adult pancreatic ductal cells in the context of oncogenic PI3K/Akt pathway activation. ResultsSimultaneous deletion of Arid1a and Pten in pancreatic ductal cells resulted in the development of ITPN, which progressed to PDAC, in mice. Simultaneous loss of Arid1a and Pten induced dedifferentiation of pancreatic ductal cells and Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) pathway activation. Consistent with the mouse data, TAZ expression was found elevated in human ITPNs and ITPN-derived PDACs but not in human intraductal papillary mucinous neoplasms, indicating that activation of the TAZ pathway is a distinctive feature of ITPN. Furthermore, pharmacological inhibition of the YAP/TAZ pathway suppressed the dedifferentiation of pancreatic ductal cells and development of ITPN in Arid1a and Pten double-knockout mice. ConclusionConcurrent loss of Arid1a and Pten in adult pancreatic ductal cells induced ITPN and ITPN-derived PDAC in mice through aberrant activation of the YAP/TAZ pathway, and inhibition of the YAP/TAZ pathway prevented the development of ITPN. These findings provide novel insights into the pathogenesis of ITPN-derived PDAC and highlight the YAP/TAZ pathway as a potential therapeutic target.

Bone Marrow-Derived Stem Cell Factor Regulates Prostate Cancer-Induced Shifts in Pre-Metastatic Niche Composition.

Skeletal metastasis is the leading cause of morbidity and mortality in prostate cancer, with 80% of advanced prostate cancer patients developing bone metastases. Before metastasis, bone remodeling occurs, stimulating pre-metastatic niche formation and bone turnover, and platelets govern this process. Stem cell factor (SCF, Kit Ligand) is increased in advanced prostate cancer patient platelet releasates. Further, SCF and its receptor, CD117/c-kit, correlate with metastatic prostate cancer severity. We hypothesized that bone-derived SCF plays an important role in prostate cancer tumor communication with the bone inducing pre-metastatic niche formation. We generated two cell-specific SCF knockout mouse models deleting SCF in either mature osteoblasts or megakaryocytes and platelets. Using two syngeneic androgen-insensitive murine prostate cancer cell lines, RM1 (Ras and Myc co-activation) and mPC3 (Pten and Trp53 deletion), we examined the role of bone marrow-derived SCF in primary tumor growth and bone microenvironment alterations. Platelet-derived SCF was required for mPC3, but not RM1, tumor growth, while osteoblast-derived SCF played no role in tumor size in either cell line. While exogenous SCF induced proangiogenic protein secretion by RM1 and mPC3 prostate cancer cells, no significant changes in tumor angiogenesis were measured by immunohistochemistry. Like our previous studies, tumor-induced bone formation occurred in mice bearing RM1 or mPC3 neoplasms, demonstrated by bone histomorphometry. RM1 tumor-bearing osteoblast SCF knockout mice did not display tumor-induced bone formation. Bone stromal cell composition analysis by flow cytometry showed significant shifts in hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and osteoblast cell percentages in mice bearing RM1 or mPC3 tumors. There were no significant changes in the percentage of macrophages, osteoclasts, or osteocytes. Our study demonstrates that megakaryocyte/platelet-derived SCF regulates primary mPC3 tumor growth, while SCF originating from osteoblasts plays a role in bone marrow-derived progenitor cell composition and pre-metastatic niche formation. Further, we show that both the source of SCF and the genetic profile of prostate cancer determine the effects of SCF. Thus, targeting the SCF/CD117 signaling axis with tyrosine kinase inhibitors could affect primary prostate carcinomas or play a role in reducing bone metastasis dependent on the gene deletions or mutations driving the patients’ prostate cancer.

MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Monocyte-derived SDF1 supports optic nerve regeneration and alters retinal ganglion cells’ response to Pten deletion

Although mammalian retinal ganglion cells (RGCs) normally cannot regenerate axons nor survive after optic nerve injury, this failure is partially reversed by inducing sterile inflammation in the eye. Infiltrative myeloid cells express the axogenic protein oncomodulin (Ocm) but additional, as-yet-unidentified, factors are also required. We show here that infiltrative macrophages express stromal cell–derived factor 1 (SDF1, CXCL12), which plays a central role in this regard. Among many growth factors tested in culture, only SDF1 enhances Ocm activity, an effect mediated through intracellular cyclic AMP (cAMP) elevation and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation. SDF1 deficiency in myeloid cells (CXCL12flx/flxLysM-Cre−/+ mice) or deletion of the SDF1 receptor CXCR4 in RGCs (intraocular AAV2-Cre in CXCR4flx/flx mice) or SDF1 antagonist AMD3100 greatly suppresses inflammation-induced regeneration and decreases RGC survival to baseline levels. Conversely, SDF1 induces optic nerve regeneration and RGC survival, and, when combined with Ocm/cAMP, SDF1 increases axon regeneration to levels similar to those induced by intraocular inflammation. In contrast to deletion of phosphatase and tensin homolog (Pten), which promotes regeneration selectively from αRGCs, SDF1 promotes regeneration from non-αRGCs and enables the latter cells to respond robustly to Pten deletion; however, SDF1 surprisingly diminishes the response of αRGCs to Pten deletion. When combined with inflammation and Pten deletion, SDF1 enables many RGCs to regenerate axons the entire length of the optic nerve. Thus, SDF1 complements the effects of Ocm in mediating inflammation-induced regeneration and enables different RGC subtypes to respond to Pten deletion.

Pten heterozygosity restores neuronal morphology in fragile X syndrome mice

Genetic studies of hippocampal granule neuron development have been used to elucidate cellular functions of Pten and Fmr1. While mutations in each gene cause neurodevelopmental disorders such as autism and fragile X syndrome, how Pten and Fmr1 function alone or together during normal development is not known. Moreover, Pten mRNA is bound by the fragile X mental retardation protein (FMRP) RNA binding protein, but how this physical interaction impinges on phosphatase and tensin homolog protein (PTEN) expression is not known. To understand the interaction of PTEN and FMRP, we investigated the dentate gyrus granule neuron development in Pten and Fmr1 knockout (KO) mice. Interestingly, heterozygosity of Pten restored Fmr1 KO cellular phenotypes, including dendritic arborization, and spine density, while PTEN protein expression was significantly increased in Fmr1 KO animals. However, complete deletion of both Pten and Fmr1 resulted in a dramatic increase in dendritic length, spine density, and spine length. In addition, overexpression of PTEN in Fmr1 KO Pten heterozygous background reduced dendritic length, arborization, spine density, and spine length including pS6 levels. Our findings suggest that PTEN levels are negatively regulated by FMRP, and some Fmr1 KO phenotypes are caused by dysregulation of PTEN protein. These observations provide evidence for the genetic interaction of PTEN and FMRP and a possible mechanistic basis for the pathogenesis of Fmr1-related fragile X neurodevelopmental disorders.

The role of PTEN in primary sensory neurons in processing itch and thermal information in mice.

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Deletion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-CreER: PTENflox/flox (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4+ non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch responses are no longer present in Drg11-CreER: PTENflox/flox: TRPV1flox/flox (PTEN: TRPV1 dCKO) mice. In addition, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.