Active thymus in adult with lung cancer: preliminary results from the Adult Thymic Project.

Thymus is considered a non-functional remnant in adults, but some evidence suggest that it may harbor residual activity. Lung cancer patients represent the ideal model to study thymic residual activity, as their thymus can be easily harvested during surgery. This study was designed to confirm the presence of residual thymic activity both in adult mice (step 1) and in humans (step 2). In step 1, lung cancer was induced by activating k-ras mutation in a cohort of 20 young and adult mice. After killing, thymus and lungs were analyzed. Thymus was considered active when medullary was evident, cortico-medullary ratio was 50:50 or higher and adipose involution was present. In step 2, a cohort of 20 patients, undergoing surgery for lung cancer, had biopsy of pericardial fat pad, site of ectopic thymus. Thymus was considered present if Hassall's bodies were detected. In mice, active thymus was detected in a high proportion of cases, without significant difference between adult and young (70% vs 44.4% respectively). Two cases without evidence of lung tumor had a fully functional thymus. In humans, ectopic thymus was detected in the pericardial fat pad in 2 cases (10.5%), confirmed by immunohistochemistry. Signs of previous thymic activity were detected in 8 additional patients. Results confirmed thymus activity in animal models and humans with lung cancer, providing the rationale for future systematic mediastinal thymic biopsy. The comprehension of interactions between thymus, lymphocytes and tumor may open a new potentially targetable perspective in lung cancer.

RAS-ON inhibition overcomes clinical resistance to KRAS G12C-OFF covalent blockade.

Selective KRASG12C inhibitors have been developed to covalently lock the oncogene in the inactive GDP-bound state. Two of these molecules, sotorasib and adagrasib, are approved for the treatment of adult patients with KRASG12C-mutated previously treated advanced non-small cell lung cancer. Drug treatment imposes selective pressures leading to the outgrowth of drug-resistant variants. Mass sequencing from patients' biopsies identified a number of acquired KRAS mutations -both in cis and in trans- in resistant tumors. We demonstrate here that disease progression in vivo can also occur due to adaptive mechanisms and increased KRAS-GTP loading. Using the preclinical tool tri-complex KRASG12C-selective covalent inhibitor, RMC-4998 (also known as RM-029), that targets the active GTP-bound (ON) state of the oncogene, we provide a proof-of-concept that the clinical stage KRASG12C(ON) inhibitor RMC-6291 alone or in combination with KRASG12C(OFF) drugs can be an alternative potential therapeutic strategy to circumvent resistance due to increased KRAS-GTP loading.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

cGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is treated with ALK tyrosine kinase inhibitors (TKIs), but the lack of activity of immune checkpoint inhibitors (ICIs) is poorly understood. Here, we identified immunogenic ALK peptides to show that ICIs induced rejection of ALK+ tumors in the flank but not in the lung. A single-peptide vaccination restored priming of ALK-specific CD8+ T cells, eradicated lung tumors in combination with ALK TKIs and prevented metastatic dissemination of tumors to the brain. The poor response of ALK+ NSCLC to ICIs was due to ineffective CD8+ T cell priming against ALK antigens and is circumvented through specific vaccination. Finally, we identified human ALK peptides displayed by HLA-A*02:01 and HLA-B*07:02 molecules. These peptides were immunogenic in HLA-transgenic mice and were recognized by CD8+ T cells from individuals with NSCLC, paving the way for the development of a clinical vaccine to treat ALK+ NSCLC.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Genomic Profiling Identifies Putative Pathogenic Alterations in NSCLC Brain Metastases.

Introduction: Brain metastases (BM) severely affect the prognosis and quality of life of patients with NSCLC. Recently, molecularly targeted agents were found to have promising activity against BM in patients with NSCLC whose primary tumors carry "druggable" mutations. Nevertheless, it remains critical to identify specific pathogenic alterations that drive NSCLC-BM and that can provide novel and more effective therapeutic targets. Methods: To identify potentially targetable pathogenic alterations in NSCLC-BM, we profiled somatic copy number alterations (SCNAs) in 51 matched pairs of primary NSCLC and BM samples from 33 patients with lung adenocarcinoma and 18 patients with lung squamous cell carcinoma. In addition, we performed multiregion copy number profiling on 15 BM samples and whole-exome sequencing on 40 of 51 NSCLC-BM pairs. Results: BM consistently had a higher burden of SCNAs compared with the matched primary tumors, and SCNAs were typically homogeneously distributed within BM, suggesting BM do not undergo extensive evolution once formed. By comparing focal SCNAs in matched NSCLC-BM pairs, we identified putative BM-driving alterations affecting multiple cancer genes, including several potentially targetable alterations in genes such as CDK12, DDR2, ERBB2, and NTRK1, which we validated in an independent cohort of 84 BM samples. Finally, we identified putative pathogenic alterations in multiple cancer genes, including genes involved in epigenome editing and 3D genome organization, such as EP300, CTCF, and STAG2, which we validated by targeted sequencing of an independent cohort of 115 BM samples. Conclusions: Our study represents the most comprehensive genomic characterization of NSCLC-BM available to date, paving the way to functional studies aimed at assessing the potential of the identified pathogenic alterations as clinical biomarkers and targets.

Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non-Small Cell Lung Cancer.

PURPOSE: Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking. EXPERIMENTAL DESIGN: We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non-small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations. RESULTS: In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors. CONCLUSIONS: This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

Tyrosine phosphatases regulate resistance to ALK inhibitors in ALK+ anaplastic large cell lymphoma.

Anaplastic large cell lymphomas (ALCLs) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. Targeting ALK using tyrosine kinase inhibitors (TKIs) is a therapeutic option in cases relapsed after chemotherapy, but TKI resistance may develop. By applying genomic loss-of-function screens, we identified PTPN1 and PTPN2 phosphatases as consistent top hits driving resistance to ALK TKIs in ALK+ ALCL. Loss of either PTPN1 or PTPN2 induced resistance to ALK TKIs in vitro and in vivo. Mechanistically, we demonstrated that PTPN1 and PTPN2 are phosphatases that bind to and regulate ALK phosphorylation and activity. In turn, oncogenic ALK and STAT3 repress PTPN1 transcription. We found that PTPN1 is also a phosphatase for SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating SHP2, the MAPK, and JAK/STAT pathways. RNA sequencing of patient samples that developed resistance to ALK TKIs showed downregulation of PTPN1 and PTPN2 associated with upregulation of SHP2 expression. Combination of crizotinib with a SHP2 inhibitor synergistically inhibited the growth of wild-type or PTPN1/PTPN2 knock-out ALCL, where it reverted TKI resistance. Thus, we identified PTPN1 and PTPN2 as ALK phosphatases that control sensitivity to ALK TKIs in ALCL and demonstrated that a combined blockade of SHP2 potentiates the efficacy of ALK inhibition in TKI-sensitive and -resistant ALK+ ALCL.

Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma.

The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

Complete and prolonged response to anti-PD1 therapy in an ALK rearranged lung adenocarcinoma.

OBJECTIVE: Immune checkpoint inhibitors (ICI) have become a major treatment in advanced non small cell lung cancer (NSCLC). However, some patients do not benefit from ICI, especially those harboring an ALK rearrangement. Here, we report a case of prolonged complete tumor response to immunotherapy in an ALK-rearranged NSCLC patient. MATERIALS AND METHODS: We verify ALK expression and rearrangement on formalin-fixed paraffin-embedded tumor samples of the patient by Immunohistochemistry and Fluorescence In Situ Hybridization analysis. The patient provided written informed consent authorizing publication of clinical case. RESULTS: We report the case of 48 years old man with a ALK-rearranged NSCLC. This patient displayed a complete response for 16 months under nivolumab therapy in third line setting after ceritinib and platin based chemotherapy. CONCLUSION: This is the first case of complete and prolonged response to ICI in ALK rearranged NSCLC. This case supports the idea that some ALK rearranged NSCLC could durably benefit from immunotherapy.

Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma.

In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-ß. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.

Comment on "ALK is a therapeutic target for lethal sepsis".

Physiologically relevant ALK (anaplastic lymphoma kinase) expression was not detected in human and mouse monocytes and macrophages, suggesting that the effects of bioactive compounds on stimulator of interferon genes (STING) activation may not depend on ALK.

Tumor Resistance against ALK Targeted Therapy-Where It Comes From and Where It Goes.

Anaplastic lymphoma kinase (ALK) is a validated molecular target in several ALK-rearranged malignancies, particularly in non-small-cell lung cancer (NSCLC), which has generated considerable interest and effort in developing ALK tyrosine kinase inhibitors (TKI). Crizotinib was the first ALK inhibitor to receive FDA approval for ALK-positive NSCLC patients treatment. However, the clinical benefit observed in targeting ALK in NSCLC is almost universally limited by the emergence of drug resistance with a median of occurrence of approximately 10 months after the initiation of therapy. Thus, to overcome crizotinib resistance, second/third-generation ALK inhibitors have been developed and received, or are close to receiving, FDA approval. However, even when treated with these new inhibitors tumors became resistant, both in vitro and in clinical settings. The elucidation of the diverse mechanisms through which resistance to ALK TKI emerges, has informed the design of novel therapeutic strategies to improve patients disease outcome. This review summarizes the currently available knowledge regarding ALK physiologic function/structure and neoplastic transforming role, as well as an update on ALK inhibitors and resistance mechanisms along with possible therapeutic strategies that may overcome the development of resistance.

Beta-adrenergic regulation of the heart expressing the Ser1700A/Thr1704A mutated Cav1.2 channel.

Beta-adrenergic stimulation of the heart increases ICa. PKA dependent phosphorylation of several amino acids (among them Ser 1700 and Thr 1704 in the carboxy-terminus of the Cav1.2 α1c subunit) has been implicated as decisive for the ß-adrenergic up-regulation of cardiac ICa. Mutation of Ser 1700 and Thr 1704 to alanine results in the Cav1.2PKA_P2-/- mice. Cav1.2PKA_P2-/- mice display reduced cardiac L-type current. Fractional shortening and ejection fraction in the intact animal and ICa in isolated cardiomyocytes (CM) are stimulated by isoproterenol. Cardiac specific expression of the mutated Cav1.2PKA_P2-/- gene reduces Cav1.2 α1c protein concentration, ICa, and the ß-adrenergic stimulation of L-type ICa in CMs. Single channels were not detected on the CM surface of the cCav1.2PKA_P2-/- hearts. This outcome supports the notion that S1700/1704 is essential for expression of the Cav1.2 channel and that isoproterenol stimulates ICa in Cav1.2PKA_P2-/- CMs.

Murine cardiac growth, TRPC channels, and cGMP kinase I.

Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM mice, and trpc3(-/-)/6(-/-) × ßRM mice. ßRM mice express cGKIß only in the smooth muscle on a cGKI(-/-) background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM, and trpc3(-/-)/6(-/-) × ßRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 (-/-) mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6(-/-), minimal or absent in trpc3(-/-), moderate in ßRM, and dramatic in trpc3(-/-)/6(-/-) ßRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in ßRM mice on a TRPC3/6 × cGKI(-/-)KO background, indicating an "additive" coupling between both signaling pathways.

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis.

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIß only in smooth muscle (ßRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, ßRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged ßRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg ⋅ kg(-1) ⋅ d(-1)) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in ßRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFß, and CTGF mRNA in Ctr but not in ßRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.

cAMP-specific phosphodiesterases 8A and 8B, essential regulators of Leydig cell steroidogenesis.

Phosphodiesterase (PDE) 8A and PDE8B are high-affinity, cAMP-specific phosphodiesterases that are highly expressed in Leydig cells. PDE8A is largely associated with mitochondria, whereas PDE8B is broadly distributed in the cytosol. We used a new, PDE8-selective inhibitor, PF-04957325, and genetically ablated PDE8A(-/-), PDE8B(-/-) and PDE8A(-/-)/B(-/-) mice to determine roles for these PDEs in the regulation of testosterone production. PF-04957325 treatment of WT Leydig cells or MA10 cells increased steroid production but had no effect in PDE8A (-/-)/B(-/-) double-knockout cells, confirming the selectivity of the drug. Moreover, under basal conditions, cotreatment with PF-04957325 plus rolipram, a PDE4-selective inhibitor, synergistically potentiated steroid production. These results suggest that the pool(s) of cAMP regulating androgen production are controlled by PDE8s working in conjunction with PDE4. Likewise, PDE8A (-/-)/B(-/-) cells had higher testosterone production than cells from either PDE8A(-/-) or PDE8B(-/-) mice, suggesting that both PDE8s work in concert to regulate steroid production. We further demonstrate that combined inhibition of PDE8s and PDE4 greatly increased PKA activity including phosphorylation of cholesterol-ester hydrolase (CEH)/hormone-sensitive lipase (HSL). CEH/HSL phosphorylation also was increased in PDE8A(-/-)/B(-/-) cells compared with WT cells. Finally, combined inhibition of PDE8s and PDE4 increased the expression of steroidogenic acute regulatory (StAR) protein. Together these findings suggest that both PDE8A and PDE8B play essential roles to maintain low cAMP levels, thereby suppressing resting steroidogenesis by keeping CEH/HSL inactive and StAR protein expression low. They also suggest that in order for PDE inhibitor therapy to be an effective stimulator of steroidogenesis, both PDE8 isozymes and PDE4 need to be simultaneously targeted.

Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack expression of the protein dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies as well as cardiomyopathy, which manifests as congestive heart failure and arrhythmias. Like humans, dystrophin-deficient mice (mdx mice) show cardiac dysfunction as evidenced by a decrease in diastolic function followed by systolic dysfunction later in life. We have investigated whether sildenafil citrate (Viagra), a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction present in the mdx mice. By using echocardiography, we show that chronic sildenafil treatment reduces functional deficits in the cardiac performance of aged mdx mice, with no effect on normal cardiac function in WT controls. More importantly, when sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. It is recognized that PDE5 inhibitors can have cardioprotective effects in other models of cardiac damage, but the present study reports a prevention and reversal of pathological cardiac dysfunction as measured by functional analysis in a mouse model of DMD. Overall, the data suggest that PDE5 inhibitors may be a useful treatment for the cardiomyopathy affecting patients with DMD at early and late stages of the disease.

Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes.

In ventricular myocytes, activation of protein kinase A (PKA) by 3'-5' cyclic adenosine monophosphate (cAMP) increases the force of contraction by increasing L-type Ca(2+) channel currents (I(Ca)) and sarcoplasmic reticulum (SR) Ca(2+) release during excitation-contraction coupling. Cyclic-nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes whose role in the cell is to regulate the spatial and temporal profile of cAMP signals by controlling the degradation of this second messenger. At present, however, the molecular identity and functional roles of the PDEs expressed in ventricular myocytes are incompletely understood. Here, we tested the hypothesis that PDE8A plays a critical role in the modulation of at least one compartment of cAMP and hence PKA activity during beta-adrenergic receptor (betaAR) activation in ventricular myocytes. Consistent with this hypothesis, we found that PDE8A transcript and protein are expressed in ventricular myocytes. Our data indicate that evoked [Ca(2+)](i) transients and I(Ca) increased to a much larger extent in PDE8A null (PDE8A(-/-)) than in wild-type (WT) myocytes during beta-adrenergic signaling activation. In addition, Ca(2+) spark activity was higher in PDE8A(-/-) than in WT myocytes. Our data indicate that PDE8A is a novel cardiac PDE that controls one or more pools of cAMP implicated in regulation of Ca(2+) movement through cardiomyocyte.