PTEN-deficient cancers depend on PIK3CB Wee, Susan; Wiederschain, Dmitri; Maira, Sauveur-Michel ...
Proceedings of the National Academy of Sciences - PNAS,
09/2008, Letnik:
105, Številka:
35
Journal Article
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Deregulation of the PI3K signaling pathway is observed in many human cancers and occurs most frequently through loss of PTEN phosphatase tumor suppressor function or through somatic activating ...mutations in the Class IA PI3K, PIK3CA. Tumors harboring activated p110α, the protein product of PIK3CA, require p110α activity for growth and survival and hence are expected to be responsive to inhibitors of its lipid kinase activity. Whether PTEN-deficient cancers similarly depend on p110α activity to sustain activation of the PI3K pathway has been unclear. In this study, we used a single-vector lentiviral inducible shRNA system to selectively inactivate the three Class IA PI3Ks, PIK3CA, PIK3CB, and PIK3CD, to determine which PI3K isoforms are responsible for driving the abnormal proliferation of PTEN-deficient cancers. Down-regulation of PIK3CA in colorectal cancer cells harboring mutations in PIK3CA inhibited downstream PI3K signaling and cell growth. Surprisingly, PIK3CA depletion affected neither PI3K signaling nor cell growth in 3 PTEN-deficient cancer cell lines. In contrast, down-regulation of the PIK3CB isoform, which encodes p110β, resulted in pathway inactivation and subsequent inhibition of growth in both cell-based and in vivo settings. This essential function of PIK3CB in PTEN-deficient cancer cells required its lipid kinase activity. Our findings demonstrate that although p110α activation is required to sustain the proliferation of established PIK3CA-mutant tumors, PTEN-deficient tumors are dependent instead on p110β signaling. This unexpected finding demonstrates the need to tailor therapeutic approaches to the genetic basis of PI3K pathway activation to achieve optimal treatment response.
The RAS pathway is one of the most frequently deregulated pathways in cancer. RAS signals through multiple effector pathways, including the RAF/mitogen-activated protein kinase (MAPK)/extracellular ...signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK and phosphatidylinositol 3-kinase (PI3K)-AKT signaling cascades. The oncogenic potential of these effector pathways is illustrated by the frequent occurrence of activating mutations in BRAF and PIK3CA as well as loss-of-function mutations in the tumor suppressor PTEN, a negative regulator of PI3K. Previous studies have found that whereas BRAF mutant cancers are highly sensitive to MEK inhibition, RAS mutant cancers exhibit a more variable response. The molecular mechanisms responsible for this heterogeneous response remain unclear. In this study, we show that PI3K pathway activation strongly influences the sensitivity of RAS mutant cells to MEK inhibitors. Activating mutations in PIK3CA reduce the sensitivity to MEK inhibition, whereas PTEN mutations seem to cause complete resistance. We further show that down-regulation of PIK3CA resensitizes cells with co-occurring KRAS and PIK3CA mutations to MEK inhibition. At the molecular level, the dual inhibition of both pathways seems to be required for complete inhibition of the downstream mammalian target of rapamycin effector pathway and results in the induction of cell death. Finally, we show that whereas inactivation of either the MEK or PI3K pathway leads to partial tumor growth inhibition, targeted inhibition of both pathways is required to achieve tumor stasis. Our study provides molecular insights that help explain the heterogeneous response of KRAS mutant cancers to MEK pathway inhibition and presents a strong rationale for the clinical testing of combination MEK and PI3K targeted therapies.
The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for identifying and validating cancer drug targets. Current transient ...siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively. In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression. We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines. Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner. Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent. An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases. Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo. The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.
To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models.
...Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group (
= 1) trial.
Consistent with clinical results, cetuximab was efficacious in wild-type
and
PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored
or
mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that
and
mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a
value of 0.01 compared with cetuximab alone. In 12 models with
mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44
mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition.
MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a
or
mutation.
.
Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T‐cell lymphoma (CTCL). To gain a better understanding of ...the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat‐induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat‐induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat‐induced apoptosis, constitutively activated NF‐κB and high levels of Bcl‐2 were observed. Inhibition of Bcl‐2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat‐resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti‐apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.
The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB ...pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.
The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine ...triphosphate-binding protein)-coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.
Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that ...specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.
Combinations of drug treatments based on bortezomib or lenalidomide plus steroids have resulted in very high response rates in multiple myeloma. However, most patients still relapse, indicating the ...need for novel combination partners to increase duration of response or to treat relapsed disease. We explored the antimyeloma activity of triple combinations of these well-established schemes with panobinostat, a novel deacetylase inhibitor with a multi-targeted profile.
The activity of these combinations was explored in vitro in cell lines by using MTT and annex-in V, ex vivo by flow cytometry, and in vivo using two different murine models of human myeloma: one bearing a subcutaneous plasmacytoma and another with a disseminated myeloma. Moreover, gene expression profiling and immunohistochemical studies were performed.
The addition of panobinostat (LBH589) to dexamethasone and either bortezomib or lenalidomide resulted in clear potentiation in multiple myeloma cell lines, freshly isolated plasma cells, and murine models of multiple myeloma. The quantification of the potency of these combinations by using the Chou-Talalay method showed synergistic combination indices for all of them. This effect derived from the deregulation of a cluster of genes that was completely different from the sum of genes affected by the single agents (895 and 1323 genes exclusively deregulated by panobinostat and dexamethasone plus bortezomib or lenalidomide, respectively). Functional experiments, such as annexin V staining, cell cycle analysis, and immunohistochemical studies also supported this potentiation. Anti-myeloma efficacy was confirmed in an extramedullary plasmacytoma model and a disseminated luciferized model, in which panobinostat also provided a marked benefit in bone disease.
The potent activity, together with the exclusive mechanistic profile, provides the rationale for the clinical evaluation of these drug combinations in multiple myeloma.
The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was ...identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure−activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.
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