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PDK1 L155E restores nutrient receptor expression and thymocyte differentiation. (A) A schematic representation of PDK1 L155E. A leucine (L) to glutamate (E) mutation at residue 155 in the PIF domain of PDK1 (denoted by *) prevents docking of PDK1 at the H-motif of substrates S6K, SGK and RSK. This prevents their T loop phosphorylation by PDK1 and subsequent activation. Dotted lines represent these inactivated pathways. PDK1 L155E mutation is permissive for PKB activation (solid line)(Collins et al, 2003). (B) Data show CD98 (left panel) and CD71 (right panel) expression on control T-PDK1+/− (PDK1+/flΔneoLck-Cre+) and T-PDK1L155E/− (PDK1L155E/flΔneoLck-Cre+) DN4 thymocytes. (C) Data show CD44 and CD25 expression on T-PDK1−/− (PDK1flΔneo/flΔneoLck-Cre+), T-PDK1+/− and T-PDK1L155E/− DNs. Data are representative of four independent experiments. (D) Flow cytometric analysis of CD4 and CD8 expression on T-PDK1−/−, T-PDK1+/− and T-PDK1L155E/− Thy1+ thymocytes is shown. Data are representative of four independent experiments. (E) Data show TCRβ staining of Thy1-gated T-PDK1+/− and T-PDK1L155E/− thymocytes (F) Data shows intracellular staining for phosphorylated S6 ribosomal protein in T-PDK1+/− and T-PDK1L155E/− DN4s. Data show phospho S6 staining in thymocytes immediately ex vivo (filled histogram) or treated with 20 nM rapamycin (open histogram) as an internal negative control to reduce S6 protein phosphorylation to basal levels. Data are representative of three independent experiments. (G) Data show western blot analysis of phosphoGSK3 α/β Ser21/9 and S6 ribosomal protein Ser235/6 in wild type (WT), T-PDK1L155E/− (L155E) and T-PDK1−/− DN3 and DN4 thymocytes.
Source publication
Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T...
Contexts in source publication
Context 1
... alternative and powerful strategy to explore the relative contribu- tions of different PDK1 substrates to a biological response in vivo is to analyse mice with 'knock-in' mutations of PDK1 alleles (Collins et al, 2003(Collins et al, , 2005McManus et al, 2004;Bayascas et al, 2006). One such PDK1 mutant, containing a L155E mutation, cannot support activation of S6K and RSK but allows activation of PKB (Collins et al, 2003; Figure 3A). ...Context 2
... that are homozygous for the PDK1 L155E mutation do not survive beyond developmental stage E12. However, it is possible to generate thymocytes that selectively express PDK1 L155E (T-PDK1 L155E/À ) by backcrossing mice that express a single PDK1 L155E allele and a single PDK1 floxed allele (PDK1 L155E/flDneo ) with mice expressing Cre recombinase under the control of the p56 lck proximal promoter (Supplementary Figure 3). The presence of the single PDK1 floxed allele in all tissues allows normal mouse development. ...Context 3
... we compared CD71 and CD98 expression in DN4 thymocytes from T-PDK1 þ /À and T-PDK1 L155E/À mice. Figure 3B shows that expression of PDK1 L155E restores CD71 and CD98 expression to a level comparable to that seen in DN4s expressing WT PDK1. PDK1 L155E could also restore thymocyte differentiation in PDK1 null pre-T cells. ...Context 4
... L155E could also restore thymocyte differentiation in PDK1 null pre-T cells. The distribution of the different DN subpopulations in T-PDK1 þ /À and T-PDK1 L155E/À mice was comparable ( Figure 3C). Figure 3D shows that unlike T-PDK1 À/À thymi, which have virtually no DPs and single positives (SPs), T-PDK1 L155E/À thymi contain DP and SP subsets at relatively normal ratios. ...Context 5
... distribution of the different DN subpopulations in T-PDK1 þ /À and T-PDK1 L155E/À mice was comparable ( Figure 3C). Figure 3D shows that unlike T-PDK1 À/À thymi, which have virtually no DPs and single positives (SPs), T-PDK1 L155E/À thymi contain DP and SP subsets at relatively normal ratios. Mature WT SP thymocytes express high surface levels of the TCRb and there was a normal frequency of these cells in T-PDK L155E/À thymi ( Figure 3E). ...Context 6
... 3D shows that unlike T-PDK1 À/À thymi, which have virtually no DPs and single positives (SPs), T-PDK1 L155E/À thymi contain DP and SP subsets at relatively normal ratios. Mature WT SP thymocytes express high surface levels of the TCRb and there was a normal frequency of these cells in T-PDK L155E/À thymi ( Figure 3E). PDK1 L155E can thus substitute for PDK1 loss and is sufficient for normal CD71 and CD98 expression and for pre-T cell differentiation into DPs and SPs. ...Context 7
... levels of S6 phosphorylation are high in WT DN4s, but absent following PDK1 deletion ( Hinton et al, 2004). Figure 3F shows that expression of a single WT PDK1 allele can support activation of S6K1 in pre-T cells, but the PDK1 L155E allele cannot. These data were confirmed by western blot analysis ( Figure 3G). ...Context 8
... 3F shows that expression of a single WT PDK1 allele can support activation of S6K1 in pre-T cells, but the PDK1 L155E allele cannot. These data were confirmed by western blot analysis ( Figure 3G). Western blot data also confirm that PDK1 L155E can support PKB activity as determined by the normal phosphorylation of GSK3a on its PKB substrate sequence Serine 21 in both WT and PDK1 L155E pre-T cells ( Figure 3G). ...Context 9
... data were confirmed by western blot analysis ( Figure 3G). Western blot data also confirm that PDK1 L155E can support PKB activity as determined by the normal phosphorylation of GSK3a on its PKB substrate sequence Serine 21 in both WT and PDK1 L155E pre-T cells ( Figure 3G). In contrast, GSK3a Serine 21 phosphorylation is absent in PDK1-null pre-T cells. ...Citations
... Previous studies have shown that proliferation but not survival of DN4 cells is dependent on IL-7R signaling, which functions to repress Bcl6 expression 10 . Similarly, proliferation during this stage of development also requires the combined activities of NOTCH and pre-TCR signaling [11][12][13][14] . This effect is in part the result of induction of Fbxl1 and Fbxl12, which induce polyubiquitination and proteasomal degradation of Cdkn1b, thereby ensuring proper cell cycle progression and proliferation 15 . ...
Production of a functional peripheral T cell compartment typically involves massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment of double-negative (DN) 2 cells and after successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes the transition of DN4 cells to the DP stage. The signals driving the expansion of DN2 thymocytes are well studied. However, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2, which suppresses cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor regulating the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway in driving apoptosis of developing T cells.
... The PI3K-phosphoinositol-dependent protein kinase-1 (PDK1)-Akt axis plays a critical role in thymocyte maturation (55,56). In addition to their well-described role in protein synthesis via mTOR signaling, PI3K dominates aerobic glycolysis and glucose metabolism in a variety of biological processes (57,58). ...
... In addition to their well-described role in protein synthesis via mTOR signaling, PI3K dominates aerobic glycolysis and glucose metabolism in a variety of biological processes (57,58). PDK1 regulates the expression of key amino acid and iron transporters and controls the switch of glucose metabolism from aerobic oxidation to glycolysis in the thymus (55,59). Loss of PDK1 impairs nutrient receptor expression and hence renders metabolically deficient to meet the energy demands from the DN to DP stage transition (55,60). ...
... PDK1 regulates the expression of key amino acid and iron transporters and controls the switch of glucose metabolism from aerobic oxidation to glycolysis in the thymus (55,59). Loss of PDK1 impairs nutrient receptor expression and hence renders metabolically deficient to meet the energy demands from the DN to DP stage transition (55,60). Furthermore, Akt signaling is a major stimulus of anabolism (21, 61,62). ...
T cell development in the thymus is tightly controlled by complex regulatory mechanisms at multiple checkpoints. Currently, many studies have focused on the transcriptional and posttranslational control of the intrathymic journey of T-cell precursors. However, over the last few years, compelling evidence has highlighted cell metabolism as a critical regulator in this process. Different thymocyte subsets are directed by distinct metabolic pathways and signaling networks to match the specific functional requirements of the stage. Here, we epitomize these metabolic alterations during the development of a T cell and review several recent works that provide insights into equilibrating metabolic quiescence and activation programs. Ultimately, understanding the interplay between cellular metabolism and T cell developmental programs may offer an opportunity to selectively regulate T cell subset functions and to provide potential novel therapeutic approaches to modulate autoimmunity.
... The competitive PDPK1 inhibitor and orphan drug OSU-03012 (AR-12) was another targeted drug with lineage specificity (Fig. 5e), where our results indicated excellent sensitivity in most T-ALL cell lines and in a subset of BCP-ALL cell lines. This could reflect lineage-related biology of the PDPK1 kinase, which mediates NOTCH1 signaling during pre-T-cell development 74 . ...
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/forall .
... Cd8b1 that codes for the b chain of CD8ab heterodimers started to be expressed in DN4 cd À that are the direct precursors of immature single-positive (ISP) cells, an intermediate CD8 + CD4 À stage bridging the DN4 and CD8 + CD4 + DP stages. Consistent with their robust proliferation, DN3b cd + , DN3b cd À , and DN4 cd À cells expressed Tfrc transcripts that code for the transferrin receptor (also known as CD71; Kelly et al, 2007). As expected, expression of Cd24 persisted in DN3b cd À and DN4 cd À cells and started decreasing in immature DN4 cd + prior to their differentiation into peripheral mature CD24 À TCRcd + cells (Parker & Ciofani, 2020). ...
After entering the adult thymus, bipotent T-cell progenitors give rise to αβ or γδ T cells. To determine whether the γδ T-cell receptor (TCR) has an instructive role in γδ T-cell lineage commitment or only "confirms" a pre-established γδ Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for γδ TCR signaling. Although these mice showed a T-cell development block at the CD4- CD8- double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of γδ TCR (further referred to as γδint ) at their surface. Single-cell transcriptomics of LAT-deficient DN3 γδint cells demonstrated no sign of commitment to the γδ T-cell lineage, apart from γδ TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 γδint cells were actively proliferating and progressed up to the DN4 stage. However, even those cells failed to turn on the transcriptional program associated with the γδ T-cell lineage. Therefore, the γδ TCR-LAT signaling axis builds upon a γδ T-cell uncommitted lineage state to fully instruct adult γδ T-cell lineage specification.
... Apart from the effect of PDK1 deletion on T cell lineage development [64,65], PDK1 was further proved to overwhelm metastasis of BC by regulating the immune cells of TME in a PyMT-induced BC mice model by mediating macrophage polarization [49,66]. Myeloid-specific inactivation of PDK1 reprogrammed the metabolism of tumor-infiltrating macrophages and stimulated M1 macrophage polarization by inhibiting the mTOR pathway, while also retarding tumor growth and suppressing lung metastasis of BC model [66]. ...
... It is imperative to develop more innovative options for a targeted PDK1 treatment strategy in BCs. As noted above, the involvement of PDK1 in T cell lineage development [64,65] makes it possible to combine PDK1-targeted therapy with immunotherapy in BCs, such as immune checkpoint inhibitors (ICIs) and anti-PD1/PDL1 therapy. The clinical feasibility, safety, and potential effectiveness of combining PDK1 inhibitors in anti-PD1/PDL1-based drug therapy in BCs deserve further verification, which might lead to breakthroughs in BC treatment. ...
Simple Summary
Approximately 2,261,419 new cases of breast cancer (BC) and 684,996 BC-related deaths are estimated to occur in 2020 globally. New individualized therapeutic strategies are urgently needed for affected patients. The aim of our review was to assess the potential role of targeted PDK1 therapies in BC. We hope the information provided on clinical trials of PDK1-targeted therapies will benefit researchers and clinicians in the breast cancer field.
Abstract
Given that 3-phosphoinositide-dependent kinase 1 (PDK1) plays a crucial role in the malignant biological behaviors of a wide range of cancers, we review the influence of PDK1 in breast cancer (BC). First, we describe the power of PDK1 in cellular behaviors and characterize the interaction networks of PDK1. Then, we establish the roles of PDK1 in carcinogenesis, growth and survival, metastasis, and chemoresistance in BC cells. More importantly, we sort the current preclinical or clinical trials of PDK1-targeted therapy in BC and find that, even though no selective PDK1 inhibitor is currently available for BC therapy, the combination trials of PDK1-targeted therapy and other agents have provided some benefit. Thus, there is increasing anticipation that PDK1-targeted therapy will have its space in future therapeutic approaches related to BC, and we hope the novel approaches of targeted therapy will be conducive to ameliorating the dismal prognosis of BC patients.
... The expression of CD98 (a subunit of the neutral amino acid transporter), CD71 (the transferrin receptor), and CD40L in these 6PGD-deficient Tregs was enhanced ( Figure 2C). CD71 and CD98 are key nutrient receptors in Tregs that depend on mTORC1 (Kelly et al., 2007). ...
Cellular metabolism has key roles in T cells differentiation and function. CD4 ⁺ T helper-1 (Th1), Th2, and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis. Here, we showed that blocking 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shifts toward Th1, Th2, and Th17 phenotypes which led to the development of fetal inflammatory disorder in mice model. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD blocked Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.
... Apart from the effect of PDK1 deletion on T cell lineage development [64,65], PDK1 was further proved to overwhelm metastasis of BC by regulating the immune cells of TME in a PyMT-induced BC mice model by mediating macrophage polarization [49,66]. Myeloid-specific inactivation of PDK1 reprogrammed the metabolism of tumor-infiltrating macrophages and stimulated M1 macrophage polarization by inhibiting the mTOR pathway, while also retarding tumor growth and suppressing lung metastasis of BC model [66]. ...
... It is imperative to develop more innovative options for a targeted PDK1 treatment strategy in BCs. As noted above, the involvement of PDK1 in T cell lineage development [64,65] makes it possible to combine PDK1-targeted therapy with immunotherapy in BCs, such as immune checkpoint inhibitors (ICIs) and anti-PD1/PDL1 therapy. The clinical feasibility, safety, and potential effectiveness of combining PDK1 inhibitors in anti-PD1/PDL1-based drug therapy in BCs deserve further verification, which might lead to breakthroughs in BC treatment. ...
Given that 3-Phosphoinositide-dependent kinase 1 (PDK1) plays a crucial role in malignant biological behaviors of a wide-range of cancers, we further review the influence of PDK1 in breast cancer (BC). First, we describe the power of PDK1 in cellular behaviors and extensively demonstrate the interacting networks of PDK1 via PI3K-dependent/ PI3K-independent pathway. Then we enlighten the roles of PDK1 in carcinogenesis, growth and survival, metastasis, and chemoresistance in BC cells. More important, we sort the current preclinical or clinical trials of PDK1 targeted therapy in BC and find that even though at present no selective PDK1 inhibitor is available for BC therapy, but the combination trials of PDK1 targeted therapy and other agents have demonstrated some benefit. Thus, there is increasing anticipations that PDK1 targeted therapy will have its space in future therapeutic concepts of BC, and we hope to feature PDK1 in BC to the clinic and bring the new promising to patients for targeted therapies.
... The loss of PDK1 results in a block at the DN3 to DP transition [146]. PDK1 promotes the expression of key nutrient receptors such as CD71 (transferrin receptor) and CD98 (subunit of L-amino acid transporters) [147]. PDK1 mediates Notch signals and is essential for trophic and proliferative responses in thymocytes. ...
... Interestingly, mTOR kinase activity is not required for RSK regulation, instead mTORC2 could serve as a scaffold to modulate RSK activity or targets. Pharmacological inhibition of RSK using BI-D1870, which inhibits all four RSK isoforms does not prevent the DN to DP differentiation but blocks the Notch-induced proliferative expansion of these pre-T cells [147]. Genetic studies to ablate different RSK isoforms in thymocytes should provide better insights on their role in early T cell ontogeny. ...
The mechanistic target of rapamycin (mTOR) controls cell fate and responses via its functions in regulating metabolism. Its role in controlling immunity was unraveled by early studies on the immunosuppressive properties of rapamycin. Recent studies have provided insights on how metabolic reprogramming and mTOR signaling impact peripheral T cell activation and fate. The contribution of mTOR and metabolism during early T-cell development in the thymus is also emerging and is the subject of this review. Two major T lineages with distinct immune functions and peripheral homing organs diverge during early thymic development; the αβ- and γδ-T cells, which are defined by their respective TCR subunits. Thymic T-regulatory cells, which have immunosuppressive functions, also develop in the thymus from positively selected αβ-T cells. Here, we review recent findings on how the two mTOR protein complexes, mTORC1 and mTORC2, and the signaling molecules involved in the mTOR pathway are involved in thymocyte differentiation. We discuss emerging views on how metabolic remodeling impacts early T cell development and how this can be mediated via mTOR signaling.
... DN3e were therefore the most immature thymocyte subset detected in thymus autonomy ( Figure 4B). To characterize the DN3 further, we assessed the expression of surface markers associated with b-selection, namely, the co-stimulatory molecules CD27 and CD28 and the transporters CD71 and CD98 (Kelly et al., 2007;Taghon et al., 2006;Williams et al., 2005). In thymus autonomy, the expression pattern of CD27 and CD28 in DN3e was similar to that in the steady state ( Figures S6A and S6C). ...
T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate in vivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the β-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate in vivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.
... Notch ligation additionally triggers activation of Phosphatidylinositol-Kinase 1 (PDK-1) and important AGC-serine kinases, thereby allowing for extensive proliferation of selected T cells. 31,32 After successfully passing the ß-selection checkpoint, the cells downregulate CD25 (DN4) and subsequently transit into the double-positive (DP) stage, hallmarked by simultaneous surface expression of CD4 and CD8. Finally, mature CD4 or CD8 single-positive (SP) T cells are able to exit the thymus. ...
Intracellular adaptor proteins are indispensable for the transduction of receptor‐derived signals, as they recruit and connect essential downstream effectors. The SLy/SASH1‐adaptor family comprises three highly homologous proteins, all of them sharing conserved structural motifs. The initial characterization of the first member SLy1/SASH3 (SH3 protein expressed in lymphocytes 1) in 2001 was rapidly followed by identification of SLy2/HACS1 (hematopoietic adaptor containing SH3 and SAM domains 1) and SASH1/SLy3 (SAM and SH3 domain containing 1). Based on their pronounced sequence similarity, they were subsequently classified as one family of intracellular scaffold proteins. Despite their obvious homology, the three SLy/SASH1‐members fundamentally differ with regard to their expression and function in intracellular signaling. On the contrary, growing evidence clearly demonstrates an important role of all three proteins in human health and disease. In this review, we systematically summarize what is known about the SLy/SASH1‐adaptors in the field of molecular cell biology and immunology. To this end, we recapitulate current research about SLy1/SASH3, SLy2/HACS1, and SASH1/SLy3, with an emphasis on their similarities and differences.
