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Domrachev B, Singh S, Li D, Rudloff U. Mini-Review: PDPK1 (3-phosphoinositide dependent -1), An Emerging Stem Cell Target. J Cancer Treatment Diagn.(2021);5(1):30-35 Journal of Cancer Treatment and Diagnosis

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Mini-Review: PDPK1 (3-phosphoinositide dependent -1), An Emerging Cancer Stem Cell Target Bogdan Domrachev1, Sitanshu Singh1, Dandan Li2, Udo Rudloff1,2* 1Rare Tumor Initiative, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 2Thoracic & GI Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

Article Info ABSTRACT

Article Notes Cancer stem cells (CSCs) are subpopulations of tumor cells that possess Received: February 24, 2021 abilities for self-renewal, differentiation, and tumor initiation. Theserare Accepted: April 12, 2021 but therapy-recalcitrant cells are assumed to repopulate tumors following *Correspondence: administration of systemic chemotherapy driving therapy failure, tumor *Correspondence to: Dr. Udo Rudloff, MD PhD, Investigator, recurrence, and disease progression. In early clinical trials, anti-CSC therapies Rare Tumor Initiative Pediatric Oncology Branch, NCI Hatfield have found limited success to-date possibly due to the inherent heterogeneity Center, Center for Cancer Research – 2B-34D, 10 Center Drive, and plasticity of CSCs and the incomplete characterization of essential CSC Bethesda, MD 20892-0001, USA; Telephone No: 240-760-6238; targets. Here, we review the role of 3-phosphoinositide dependent protein E-mail: [email protected]. kinase-1 (PDPK1) as an emerging CSC target. While most previous studies have © 2021 Rudloff U. This article is distributed under the terms of relied on CSC models which are based on lineage and tissue-specific marker the Creative Commons Attribution 4.0 International License. profiles to define the relationships between putative target and CSC traits, this review discusses PDPK1 and its role in CSC biology with an emphasis on CSC systems which are based on proposed function like label-retaining cancer cells (LRCCs). Keywords: Cancer Stem Cells 3-Phosphoinositide Dependent Protein Kinase-1 The cancer stem cell (CSC) hypothesis Label Retaining Cancer Cells Drug Resistance The near universal development of resistance to systemic Chemotherapy cytotoxic chemotherapy has been a stubbornly incalcitrant problem in oncology for decades. Commonly, after an initial period of tumor

experience disease progression, detoriation of their quality ofregression life, and succumb and disease to their control illness patients1. Thus, the afflicted introduction by advanced of the CSC hypothesis, which provided a fundamentally novel explanation for the commonly observed treatment failures and raised prospects to overcome therapy resistance via novel therapy approaches, was greeted with great interest2.

within hematological malignancies at the Princess Margaret CancerThree Centre decades in Toronto, ago, the Canada identification3,4, and offrom tumor-initiating breast cancer cells by Clark and colleagues thereafter5, suggested a hierarchical model of carcinogenesis6. Contrary to the clonal evolution model which suggests stochastic cancer cell divisions resulting in biologically equivalent cancer cells in each daughter cell generation, the CSC theory holds that (1) cancer arises from cells with dysregulated

heterogeneous mass of cells which include a small fraction of self-renewal mechanisms, and (2) cancer is comprised of a these cells are functionally distinct from the bulk, differentiated cancerstem-like cells progenitor2,7,8. While cellsthe exact that driveorigin tumor of the progression,CSC remains anddebated that with experimental research supporting both CSCs arising via malignant transformation from normal stem cells as well as via

Page 30 of 35 Domrachev B, Singh S, Li D, Rudloff U. Mini-Review: PDPK1 (3-phosphoinositide dependent protein kinase-1), An Emerging Cancer Stem Cell Target. J Cancer Treatment Journal of Cancer Treatment and Diagnosis Diagn.(2021);5(1):30-35

9,10, the concept of CSCs adapting their phenotype under chemotherapy pressure is does explain the inherent heterogeneity of tumors and the intriguingly also discussed in the origin and initial emerge commonlyde-differentiation observed of cancerresistance cells to chemoradiotherapy2,11 of CSCs20

is drug resistant. Recent duefindings to the show induction that therapeutic of EMT factors pressure and chemotherapyFailure to eradicate treatment thesewith new self-renewing, cancer cells from resistant this can induce in non-CSCs a transient, stem-like state which poolcells of leads cells and to repopulationis cause for tumor of residual recurrence tumors (Fig. 1A) post-12. revert to their original phenotype upon withdrawal of However, accepting an exclusively hierarchical model to chemotherapyother CSC traits. underscoring These therapy-induced the dynamic nature CSC-like of their cells capture cancer pathogenesis is at odds with several clinical adaptive mechanisms in response to cancer therapy. burden reduction due to the assumed chemosensitivity of Intratumoral CSC heterogeneity findings: for most cancers, the concept of effective tumor CSC heterogeneity is, in large, also the result of the the clinic as tumor, in particular solid organ malignancies, complex dynamic interplay and interdependence between notthe bulkuncommonly non-CSC tumordo not cell regress population at all is uponnot observed treatment in CSCs and surrounding stromal milieu forming the CSC with systemic chemotherapy1 niche. One of the best investigated CSC niche–CSC interface are adaptations of CSCs residing in hypoxic areas21. tumor cell progeny does not apply. The when concept tumor of recurrencetreatment- Hypoxic, poorly perfused areas of solid organ cancers are occursescaping very CSCs, rapidly, self-renewal or when and cancer repopulating treatment proliferating accelerates most heavily populated with CSCs. Within these hypoxic tumor growth. The hierarchical model works best in clinical scenarios which initially see a reduction in tumor like tumor associated macrophages, suppressing dendritic burden and then, after a latency period, tumors recur. andniches cytotoxic CSCs create T acell unique function, environment upregulate by attracting inhibitory M2- 21,22. CSC Mechanisms of cancer stem cell therapy resistance – inherent versus acquired? immune checkpoints or pro-angiogenic signals ratesresiding and in shape peri-vascular vascularization regions onand the ECM other formation hand find23. patients’ tumoral biopsies support that CSCs are inherently, CSCsa nutrient-rich within the invasive environment, front of have tumors higher have proliferativeupregulated a prioriA plethora, resistant of into vitro,chemoradiotherapy in vivo as well8,14 . asInherent findings CSC in migratory and invasive capabilities, and the overlap with characteristics mediating resistance to chemoradiotherapy include upregulation of DNA damage repair mechanism programs essential for metastasis like21 e.g. the CXCR4/ on multiple levels including upregulation of G1/M and in the metastatic spread of cancers . Overall, while the CXLX12 axis suggests these cells to be causally involved G2 dynamic interplay of CSCs with nearly all stromal elements /S checkpoints, efficient scavenging of reactive oxygen ECM‘niche components,concept’ is a simplificationit provides functionalof the very rationalecomplex andfor species (ROS), increased drug efflux via upregulation of the phenotypic differences and heterogeneity observed ATP-binding cassettes and8 other drug transporters, or L . Conventional chemotherapies upregulationare most effective of pro-survival, in rapidly anti-apoptosisproliferating cells regulators going they become major educators of their microenvironment. likefrequently Bcl-2 through or Bcl-X S phase. CSCs on the other hand are Reciprocally,among CSCs. Asthese CSCs educated need to stromal find their cells niches, signal vice back versa, and known to be quiescent cells which have left the cell cycle and thus escape the genotoxic effect of many currently therapy resistance21. employed chemotherapy agents15,16. Additionally, CSCs impact CSC traits like CSC quiescence, self-renewal, or It is also likely that cancer stem cells are coming in can effectively sense chemotherapy agents as xenobiotics and out of the CSC pools increasing heterogeneity and and through activation of ALDH1A1 or ALDH3A1 pleiotropism of CSC traits within tumors further9. In which effectively can metabolize chemotherapy agents17. this regard, targeting rare, infrequent CSC populations However, CSCs are also able to acquire under therapy pressure novel therapy resistance traits18. Following on such markers to capture CSC populations mediating short courses of chemotherapy the fraction of CSCs to chemotherapydefined by CSC resistance surface markersmight have and led an to overreliance the largely

14 dramatically 7 non-CSC tumoral bulk cells has been shown to change far . Thus, there has been a renewed refocus on preclinical disappointingCSC models with early improved clinical recapitulationresults of anti-CSC of tumoral therapy stem so acquired a more. Comparing aggressive CSCs phenotype between chemotherapy-manifested by naïve versus chemotherapy-resistant conditions, CSCs cell biology, like LRCCs, which are less biased by culture conditions, cell lineage markers, or results derived from immune compromised mouse models9. theseupregulation stemness of regulators the self-renewal differed factorsbetween NANOG, administered OCT4, and SOX2 as well as of ALDH3A1. Interestingly, effects on Here we discuss the role of PDPK1 as an emerging chemotherapeutics. The significant plasticity of CSCs

Page 31 of 35 Domrachev B, Singh S, Li D, Rudloff U. Mini-Review: PDPK1 (3-phosphoinositide dependent protein kinase-1), An Emerging Cancer Stem Cell Target. J Cancer Treatment Journal of Cancer Treatment and Diagnosis Diagn.(2021);5(1):30-35

Figure 1. Role of CSCs in mediation of therapy resistance and tumor recurrence. A. Self-renewal of therapy-resistant CSCs repopulate tumors leading to therapy failure including cancer recurrence and disease progression. B. PDPK1 signaling involved in CSC function. CSC traits supported by PDPK1 are listed.

si of PDPK1 substrate explain the heterogenous and studiesregulator in ofautochthonous cancer stemness animal by reviewing models of recentcancer, findings as well pleiotropicgnaling output. PDPK1 Thusoutput the across varying different pre-activation cell lineages states and asin functionalCSC traits supportedCSC models by like PDPK1 label-retaining (Fig. 1B). cells (LRCCs), the not uncommonly observed lack of correlation of PDPK1 activity and AKT activation29,30. Regulation of cancer stemness pathways by PDPK1 Considering the pleiotropism of PDPK1 output, it is not PDPK1 is a phylogenetically conserved member surprising that PDPK1 was found to be an essential regulator and master regulator of the large AGC kinase family24. of cancer stem cell . Best examined is the Hippo signaling pathway31 PDPK1 forms in the cytoplasm a complex with Sav1 PDPK1 possesses a N-terminal kinase and a C-terminal which allows the Hippo pathway; in its components non-activated MST1/2 form, pleckstrin homology (PH) domain which senses PI3K- and LATS to phosphorylate YAP retaining this master generated phospho-inositide metabolites, in particular regulator in the cytoplasm32,33. Following kinasephosphatidylinositol domain consists (3,4,5)-trisphosphate of the activation (PtdIns(3,4,5)loop and the and membranous recruitment of PDPK1, this complex P3); PIP3), at the inner plasma membrane. The N-terminal dissolves, YAP is allowed nuclear entry and activation of to a hydrophobic motif on PDPK1 substrates25. PDPK1 the YAP/TAZ stemness program occurs. Constitutively PDPK1-interactingautophosphorylation fragment of the activation (PIF)-pocket loop which (at binds 241) activates PDKP126. Signaling output of constitutively Wnt signaling in medulloblastoma where PDPK1 small moleculeactivated PDPK1inhibition has increased been shown survival to stimulate and enhanced β-catenin/ the cytotoxic effects of chemotherapeutic drugs34 itsactivated substates PDPK1 as well is primarilyas interactions determined with other by the protein pre- mediated activation of the AGC kinase S6K1 connects activation state and post-translational modifications of PDPK1 signaling output with the hedgehog pathway. PDPK1- in suppressor candidate 4 (TUSC4) proteins24,27,28. Whereas cancer cells35. Recently, PDPK1 has also been shown to AKTmodulators activation like by thePDPK1 inhibitory is regulated 14-3-3 via or simultaneous the tumor function as an upstream regulator of the cancer stemness PIP3 binding enhancing proximity and phosphorylation master regulator Myc36 at 308 of the activation loop of AKT, other regulation of cancer stemness signaling pathways is shown AGC kinase substrates of PDPK1 like serum and in Fig. 2. . A summary of PDPK1-mediated

S6 kinase (p70S6K), (PKC), or p90RSK Cancer stem cell self-renewal and tumor initiation glucocorticoid-regulated kinase 1 (SGK1), p70 ribosomal interact with PDPK1 through their PIF-binding motifs. PLK1PDPK1 on threonine has recently 210 been which, identified once asphosphorylated, an upstream Post-translational modifications of the PIF domains of stabilizesregulator ofMyc the fromc-Myc proteasomal . PDPK1 degradation can phosphorylate via serine these substrate kinases very heavily impact the affinity of PDPK1-kinase interactions and therewith PDPK1 Page 32 of 35 Domrachev B, Singh S, Li D, Rudloff U. Mini-Review: PDPK1 (3-phosphoinositide dependent protein kinase-1), An Emerging Cancer Stem Cell Target. J Cancer Treatment Journal of Cancer Treatment and Diagnosis Diagn.(2021);5(1):30-35

Figure 2. Regulation of cancer stemness pathways by PDPK1. A. Membranous recruitment of PDPK1 (right) activates Hippo pathway. B. Activation of WNT/β-catenin signaling.C . PDPK1 activation of S6 kinase activates hedgehog pathway (HH, hedgehog; SMO, smoothened). D. PDPK1--MYC signaling axis in cancer.

62 phosphorylation36,37 always clear that increased chemoresistance is due to

. Increased cellular levels of c-Myc resultedpromote incancer loss ofcell CSCs self-renewal, and CSC functions survival, uponand increased silencing PDPK1-promoted cancer stemness. AKT signaling with ofCSC PDPK1 fractions36 in tumors as loss-of-function experiments independentits pro-survival, of tumor anti- cell differentiation functions is, state for example,and CSC function.a well-known Thus, mediator one of the of chemoresistancemore compelling in examinations cancer cells therapy36,38 . Of note, PDPK1-governed increased stability translateof c-Myc into was enhanced associated tumor with resistanceinitiation in to vivo targeted has recently been. Thatelegantly PDPK1-governed shown in autochthonous CSC function pancreas can cancerof PDPK1-governed cells (LRCCs). cancer LRCCs stemness are slowly mediating cycling, resistancequiescent cancer models. Eser and colleagues showed that hat cellsto chemotherapy holding onto emanates intracellular from dyes studies16. Slow in label-retaining cycling LRCCs PDPK1 is an essential of Kras, and that an intact exhibit cancer stem cell and pluripotency traits and PDPK1/PI3K axis is an essential tumor initiating event represent a distinct subpopulation of the heterogeneous in cooperation with KRAS for increased cell plasticity, CSC pool44,45. LRCCs have been shown to be more tumorigenic, mediate therapy resistance, and promote adenocarcinoma (PDAC) formation39 tumor recurrence15,46,47. Treatment with chemotherapy acinar-to-ductalPDPK1 promoting metaplasia tumor initiation (ADM), and in cooperationpancreatic ductal with dramatically can increase the fraction of LRCCs48. It is Erbb2 or Ras activation have been made. Similar in breast findings cancer of believed that LRCCs undergo asymmetric cell divisions cells40,41, or in BRAF V600 mutant where loss of 45. LRCCs PDPK1 reduced tumor formation42. provide a unique opportunity to study cancer stemness with non-random chromosomal co-segregation Mediation of resistance to chemotherapy While there are many studies linking PDPK1 expression retainingwithin a live,pancreas cell-based cancer system cells, Li that and reliescoworkers on stemness showed level and PDPK1 activation to chemoresistance43, it is not function rather than on marker profiles. Studying label-

that PDPK1 is significantly upregulated in LRCCs compared

Page 33 of 35 Domrachev B, Singh S, Li D, Rudloff U. Mini-Review: PDPK1 (3-phosphoinositide dependent protein kinase-1), An Emerging Cancer Stem Cell Target. J Cancer Treatment Journal of Cancer Treatment and Diagnosis Diagn.(2021);5(1):30-35

5. and response to chemotherapy treatment in LRCCs, a Al-Hajj M, Wicha MS, Benito-Hernandez A, et al: Prospective to the non-LRCC fraction and that PDPK147,49. regulatesFindings survivalthat the identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U stemness cell population survives chemotherapy and is 6. S A 100:3983-8, 2003 finding also made by another group Rich JN: Cancer stem cells: understanding tumor hierarchy and made in acute myeloid leukemia (AML) where PDPK1 7. heterogeneity. Medicine (Baltimore) 95:S2-S7, 2016 Research. J Natl Cancer Inst 108, 2016 wasthe nidus found for to re-growth be a major and regulator disease relapseof leukemia have alsostem been cell Brower V: Cancer Stem Cell Hypothesis Evolves With Emerging survival50. 8. 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Adv Exp Med Biol 1164:199-206, 2019 funds from the National Institutes of Health (NIH), and was e6, Chefetz 2019 I, Grimley E, Yang K, et al: A Pan-ALDH1A Inhibitor Induces supported by the Intramural Research Program (IRP) of the Necroptosis in Stem-like Cells. Cell Rep 26:3061-3075 18. NIH, National Cancer Institute, Center for Cancer Research and Role in Disease Progression and Therapy Resistance. Stem Cell (ZIA BC 011267) as well as donations from ‘Running for Das PK, Pillai S, Rakib MA, et al: Plasticity of Cancer Stem Cell: Origin Rachel’ and the Pomerenk family via the Rachel Guss and 19. Rev Rep 16:397-412, 2020 Bob Pomerenk Pancreas Cancer Research Fellowship to NCI. enriched in xenogeneic tumors following chemotherapy. 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