Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

Oncogenic dependency on ß-catenin in liver cancer cell lines correlates with pathway activation.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Anti-miR-21 Suppresses Hepatocellular Carcinoma Growth via Broad Transcriptional Network Deregulation.

UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.

Selective blockade of the hydrolysis of the endocannabinoid 2-arachidonoylglycerol impairs learning and memory performance while producing antinociceptive activity in rodents.

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells.

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

p600 Plays Essential Roles in Fetal Development.

p600 is a multifunctional protein implicated in cytoskeletal organization, integrin-mediated survival signaling, calcium-calmodulin signaling and the N-end rule pathway of ubiquitin-proteasome-mediated proteolysis. While push, the Drosophila counterpart of p600, is dispensable for development up to adult stage, the role of p600 has not been studied during mouse development. Here we generated p600 knockout mice to investigate the in vivo functions of p600. Interestingly, we found that homozygous deletion of p600 results in lethality between embryonic days 11.5 and 13.5 with severe defects in both embryo and placenta. Since p600 is required for placental development, we performed conditional disruption of p600, which deletes selectively p600 in the embryo but not in the placenta. The conditional mutant embryos survive longer than knockout embryos but ultimately die before embryonic day 14.5. The mutant embryos display severe cardiac problems characterized by ventricular septal defects and thin ventricular walls. These anomalies are associated with reduced activation of FAK and decreased expression of MEF2, which is regulated by FAK and plays a crucial role in cardiac development. Moreover, we observed pleiotropic defects in the liver and brain. In sum, our study sheds light on the essential roles of p600 in fetal development.

Cyclin E constrains Cdk5 activity to regulate synaptic plasticity and memory formation.

Cyclin E is a component of the core cell cycle machinery, and it drives cell proliferation by regulating entry and progression of cells through the DNA synthesis phase. Cyclin E expression is normally restricted to proliferating cells. However, high levels of cyclin E are expressed in the adult brain. The function of cyclin E in quiescent, postmitotic nervous system remains unknown. Here we use a combination of in vivo quantitative proteomics and analyses of cyclin E knockout mice to demonstrate that in terminally differentiated neurons cyclin E forms complexes with Cdk5 and controls synapse function by restraining Cdk5 activity. Ablation of cyclin E led to a decreased number of synapses, reduced number and volume of dendritic spines, and resulted in impaired synaptic plasticity and memory formation in cyclin E-deficient animals. These results reveal a cell cycle-independent role for a core cell cycle protein, cyclin E, in synapse function and memory.

Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma.

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.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

FoxM1 is a downstream target and marker of HER2 overexpression in breast cancer.

The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin-embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin-embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.

CDK4 activity in mouse embryos expressing a single D-type cyclin.

D-type cyclins (D1, D2, and D3) are components of the cell cycle machinery. Their association with cyclin-dependent kinase 4 (CDK4) and CDK6 causes activation of these protein kinases and leads to phosphorylation and inactivation of the retinoblastoma protein, pRb. Using embryos expressing single D-type cyclin ('cyclin D1-only', 'cyclin D2-only' and 'cyclin D3-only'), we tested whether each of D-type cyclin plays the same role in CDK activation and phosphorylation of pRb during mouse embryonic development. We found that the level of CDK4 activity was similar in wild-type embryos and those expressing only cyclin D3 or cyclin D2. However, we did not detect CDK4 activity in embryos expressing only cyclin D1, despite the fact that this cyclin was able to form complexes with CDK4 and p27(kip1) in wild-type as well as in mutant embryos. Analysis of the expression pattern of mRNA encoding cyclin D1 revealed that the expression of this RNA is regulated temporally during embryogenesis. These data and results from other laboratories indicate that cyclin D1-dependent CDK4 activity is dispensable for normal development of the mouse embryo.

Critical role for D-type cyclins in cellular transformation induced by E6/E7 of human papillomavirus type 16 and E6/E7/ErbB-2 cooperation.

Recently, we reported that E6/E7 of human papillomavirus (HPV) type 16 cooperates with the ErbB-2 receptor to induce cellular transformation of human normal oral epithelial (NOE) and mouse normal embryonic fibroblast (NEF) cells. Furthermore, we demonstrated that cyclin D1 is essential for this transformation induced by E6/E7 and E6/E7/ErbB-2 cooperation using cyclin D1 antisense and knockout (D1(-/-)) cells. To determine the role of all D-type cyclins (D1, D2 and D3) in E6/E7/ErbB-2 cooperation, we examined the effects of E6/E7, ErbB-2 alone and E6/E7/ErbB-2 together in NEF, NEF-D1(-/-), NEF-D2(-/-) and NEF-D3(-/-) cells. We confirm that NEF-E6/E7 and NEF-E6/E7/ErbB-2, but not NEF-ErbB-2 cells, induce colony formation in soft agar and tumor formation in nude mice. We report that E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of D1(-/-) and D2(-/-) cells in vitro and in vivo. In contrast, E6/E7/ErbB-2 together but neither E6/E7 nor ErbB-2 alone provoke cellular transformation of D3(-/-) cells. Nevertheless, D3(-/-)E6/E7/ErbB-2 cells resulted in up to a 60 and 50% decrease in colony and tumor formation in soft agar and nude mice, respectively, compared with NEF-E6/E7/ErbB-2 cells. Furthermore, using cyclin D2 small interfering RNA we inhibited tumor and colony formation of the human NOE-E6/E7-ErbB-2-transformed cell line; in contrast, cyclin D3 small interfering RNA repressed approximately 50% of colony and 40% of tumor formation of E6/E7/ErbB-2 cooperation in this cell line. These data suggest that cyclins D1, D2 and D3 (to a lesser extent) are important downstream mediators of the cellular transformation induced by E6/E7 and E6/E7/ErbB-2 cooperation in normal cells. Our data imply that anti-D-type cyclin therapies are important in the treatment of human cancers expressing high-risk HPV or HPV/ErbB-2.

Essential role for cyclin D3 in granulocyte colony-stimulating factor-driven expansion of neutrophil granulocytes.

The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G(1) cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.

Requirement for CDK4 kinase function in breast cancer.

Cyclin D1 is overexpressed in the majority of human breast cancers. We previously found that mice lacking cyclin D1 are resistant to mammary carcinomas triggered by the ErbB-2 oncogene. In this study, we investigated which function of cyclin D1 is required for ErbB-2-driven mammary oncogenesis. We report that the ability of cyclin D1 to activate cyclin-dependent kinase CDK4 underlies the critical role for cyclin D1 in breast cancer formation. We also found that the continued presence of CDK4-associated kinase activity is required to maintain breast tumorigenesis. We analyzed primary human breast cancers and found high cyclin D1 levels in a subset (approximately 25%) of ErbB-2-overexpressing tumors. We propose that this subset of breast cancer patients might benefit from inhibiting CDK4 kinase.

Ras and Myc can drive oncogenic cell proliferation through individual D-cyclins.

D-type cyclins serve as cell cycle recipients of several oncogenic pathways. The specific sequences of the promoters of the cyclin D genes are thought to render particular D-cyclins responsive to specific oncogenic pathways. For instance, the Ras oncogene was postulated to signal through cyclin D1, while Myc can impact the cell cycle machinery by transcriptionally upregulating cyclin D2. In the current study we engineered mouse fibroblasts to express only cyclin D1, only D2, or only D3. These 'single-cyclin' cells allowed us to rigorously test the ability of cyclin D1, D2, or D3, when expressed on their own, to serve as recipients of the Ras- and Myc-driven oncogenic pathways. We found that each of the D-cyclins was sufficient to drive oncogenic proliferation of mouse fibroblasts. This, together with our recent observations that cells lacking all three D-cyclins show greatly reduced susceptibility to the oncogenic action of Ras and Myc, reveals that the Ras and Myc oncogenes can impact the core cell cycle machinery through all three D-cyclins.

Mouse development and cell proliferation in the absence of D-cyclins.

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.

Cyclin D1 is essential for neoplastic transformation induced by both E6/E7 and E6/E7/ErbB-2 cooperation in normal cells.

More than 25% of head and neck squamous cell carcinomas (HNSCC) and 99% of cervical cancers (CxCa) are positive for high-risk human papillomaviruses (HPVs). Furthermore, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HNSCC and CxCa. Recently, we demonstrated that E6/E7 of HPV type 16 cooperate with ErbB-2 to induce cell transformation of human normal oral epithelial (NOE) cells. This is accompanied by overexpression of cyclin D1 in NOE cells. To determine the role of cyclin D1 in E6/E7/ErbB-2 cooperation, we examined the independent effects of E6/E7 and ErbB-2, and the combined effect of E6/E7 and ErbB-2 in mouse normal embryonic fibroblast (NEF), wild type (wt), and knockout cyclin D1 (D1(-/-)) cells. We report that NEF-wt cells transduced with E6/E7 alone and E6/E7/ErbB-2 together form small and large tumors in nude mice, respectively, as well as different sized colonies in soft agar; whereas ErbB-2 alone elicits neither tumor formation in vivo nor colony formation in soft agar. More importantly, E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of cyclin D1(-/-) cells in vivo and in vitro. Furthermore, using antisense cyclin D1 we completely inhibited tumor and colony formation of NEF-wt-E6/E7 and wt-E6/E7-ErbB-2 as well as human NOE-E6/E7-ErbB-2-transformed cells. These analyses reveal that cyclin D1 is the downstream target of the neoplastic transformation induced by E6/E7 or E6/E7/ErbB-2 cooperation in normal cells. Our data suggest that anti-cyclin D1 therapy may be highly specific in the treatment of all human cancers expressing high-risk HPVs or HPVs/ErbB-2.

Mammalian cell cycles without cyclin E-CDK2.

The family of mammalian E-type cyclins is composed of two proteins, termed cyclin E1 and E2. These two cyclins are widely expressed in proliferating cells. E-cyclins bind and activate cyclin dependent kinase CDK2. Cyclin E-CDK2 complexes were believed to play critical function in driving cell cycle progression of normal, nontransformed cells and of cancer cells. Several recent reports challenge this notion.

Cyclin E ablation in the mouse.

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

Requirement for cyclin D3 in lymphocyte development and T cell leukemias.

The D-type cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. Cyclin D3 gene is rearranged and the protein is overexpressed in several human lymphoid malignancies. In order to determine the function of cyclin D3 in development and oncogenesis, we generated and analyzed cyclin D3-deficient mice. We found that cyclin D3(-/-) animals fail to undergo normal expansion of immature T lymphocytes and show greatly reduced susceptibility to T cell malignancies triggered by specific oncogenic pathways. The requirement for cyclin D3 also operates in human malignancies, as knock-down of cyclin D3 inhibited proliferation of acute lymphoblastic leukemias deriving from immature T lymphocytes. These studies point to cyclin D3 as a potential target for therapeutic intervention in specific human malignancies.