The PI3K Pathway in Human Disease

Cutaneous melanoma has been at the forefront of targeted therapy breakthroughs over the last 5 years. The FDA approval of BRAF and MEK inhibitors for the treatment of late-stage, mutant V600E/K BRAF-harboring melanoma patients has been heralded due to high response rates and improvements in progression-free survival, but the durable effects of these inhibitors are limited. Alterations in the phosphoinositide 3-kinase (PI3K) pathway including inactivation of PTEN (a tumor suppressor that normally down-regulates PI3K signaling) and, less frequently, mutations in PIK3CA (the gene encoding the α isoform of the PI3K catalytic subunit) have been identified in mutant BRAF melanomas. Furthermore, PI3K pathway signaling is often up-regulated in association with resistance to mutant BRAF and/or MEK targeting agents. Despite the evidence highlighting the importance of the PI3K pathway in melanoma, initial testing of class I PI3K inhibitors in patients has not produced dramatic results. An issue for combinatorial studies is the overlapping toxicities of MAPK pathway inhibitors and PI3K-targeting agents that limits their effective dosing. A recent study from Deuker and colleagues provides important preclinical advances in this area by examining the PI3K isoform dependencies in mutant BRAF melanomas. PI3K functions as a heterodimer with a p110 catalytic subunit and a regulatory subunit. Four isoforms of p110 subunits exist: α, β, δ and γ (Jean and Kiger, 2014; Yuan and Cantley, 2008). p110α and p110β are ubiquitously expressed, whereas δ and γ isoforms display highest expression in immune cells. Importantly, isoform-selective PI3K inhibitors have been developed to target tumor dependencies and minimize toxicities in non-tumor cells. Deuker et al. utilized these selective inhibitors to determine the PI3K isoform dependency of mutationally active PI3Kα and PTEN-deficient subgroups of mutant BRAF melanomas using cell lines derived from mouse models and human patients. In vitro, PTEN-deficient lines were less sensitive to the α-isoform PI3K inhibitor, BYL719, compared to mutant H1047R PI3Kα cells. In contrast to other cancer types, in which PTEN-deficient tumors are often PI3Kβ-dependent (Wee et al., 2008), PTEN-null melanomas did not rely on PI3Kβ. Growth of PTEN-null mutant BRAF melanomas was blocked by a PI3Kα/δ/γ-targeting but β-sparing inhibitor, GDC-0032. In addition, the δ/γ inhibitor, IPI145, enhanced the effects of BYL719 suggesting involvement of the δ/γ isoforms in melanoma. PI3Kα-dependency is also suggested to be present in BRAF V600E/WT PTEN melanomas, which represent a substantial proportion of mutant BRAF melanomas, although confirmation is warranted in a larger panel of BRAF V600E/WT PTEN melanoma models. “More dramatic tumor regressions were observed in vivo with the combination of BRAF-MEK pathway and PI3K inhibitors. ” A long-term limitation to both BRAF and MEK inhibitors in melanoma is acquired drug resistance and patients who develop resistance have few second-line treatment options. In addition to up-front combination, treatment of MEK inhibitor-resistant tumors with the PI3K β-sparing inhibitor resulted in a cytostatic response. These data were generated from a single MEK inhibitor-resistant tumor. As multiple mechanisms of resistance are likely to occur, it will be important to further explore effects in cultured- and patient-derived BRAF and MEK inhibitor-resistant models. Overall, the study presented by Deuker et al., provides an important preclinical advance in targeting the PI3K pathway in both first-line combinations as well as a possible second-line salvage option for BRAF-MEK pathway resistant melanomas. These findings should propel investigation of methods to quantitatively and non-invasively measure PI3K signaling in preclinical in vivo melanoma models and to determine the most effective dosing regimens. Given the high degree of immune reactivity of melanomas and remarkable advances in immune-based therapies, other key issues will be to determine the effect of targeting PI3K on infiltration of immune cells into melanomas and the extent to which PI3K-targeting agents can be combined with immune-based therapies. Advances in mutant BRAF melanomas will lead to studies addressing the importance of PI3K isoform specific targeting in wild-type BRAF subsets of melanoma for which there is an unmet need for new therapeutic strategies.

Pathways activated downstream of constitutively active phosphatidylinositol (PI) 3-kinase in PTEN-deficient prostate cancer (PCa) cells are possible therapeutic targets. We found that the nonreceptor Tec family tyrosine kinase Bmx/Etk was activated by tyrosine phosphorylation downstream of Src and PI 3-kinase in PTEN-deficient LNCaP and PC3 PCa cells and that Bmx down-regulation by short interfering RNA markedly inhibited LNCaP cell growth. Bmx also associated with ErbB3 in LNCaP cells, and heregulin-beta1 enhanced this interaction and further stimulated Bmx activity. Epidermal growth factor (EGF) similarly stimulated an interaction between Bmx and EGF receptor and rapidly increased Bmx kinase activity. Bmx stimulation in response to heregulin-beta1 and EGF was Src-dependent, and heregulin-beta1 stimulation of Bmx was also PI 3-kinase-dependent. In contrast, the rapid tyrosine phosphorylation and activation of Bmx in response to EGF was PI 3-kinase-independent. Taken together, these results demonstrate that Bmx is a critical downstream target of the constitutively active PI 3-kinase in PTEN-deficient PCa cells and further show that Bmx is recruited by the EGF receptor and ErbB3 and activated in response to their respective ligands. Therefore, Bmx may be a valuable therapeutic target in PCa and other epithelial malignancies in which PI 3-kinase or EGF receptor family pathways are activated.

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

The phosphoinositide-3 kinase (PI3K) pathway has been identified as an important target in breast cancer research for a number of years, but is new to most clinicians responsible for the daily challenges of breast cancer management. In fact, the PI3K pathway is probably one of the most important pathways in cancer metabolism and growth. Mutations in the PI3K pathway are frequent in breast cancer, causing resistance to human epidermal growth factor receptor 2-targeted agents and, possibly, to hormonal agents as well. Available agents that affect the PI3K pathway include monoclonal antibodies and tyrosine kinase inhibitors, as well as PI3K inhibitors, Akt inhibitors, rapamycin analogs, and mammalian target of rapamycin (mTOR) catalytic inhibitors. Multiple PI3K inhibitors are currently under development, including pure PI3K inhibitors, compounds that block both PI3K and mTOR (dual inhibitors), pure catalytic mTOR inhibitors, and inhibitors that block Akt. It is likely that these agents will have to be given in combination with other signal inhibitors because anti-mTOR agents and PI3K inhibitors may result in the activation of compensatory feedback loops that would in turn result in decreased efficacy. This article reviews current data related to the PI3K pathway, its role in breast cancer, the frequency with which PI3K is aberrant in breast cancer, and the potential clinical implications of using agents that target the PI3K pathway.

Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor with a devastating 5-year survival rate of 6.8%. The current standard of care, consisting of surgery, radiation, and temozolomide (TMZ), has remained unchanged for over the past 15 years despite its limited efficacy and the serious therapy-related adverse events associated to this regimen. Although some frequent genetic alterations have been identified in GBM, no targeted therapies have shown efficacy in these patients to date. Among these, the phosphoinositide 3-kinase (PI3K) pathway is a signaling network that regulates cell growth, proliferation, migration, and invasion. Extensive molecular characterization of GBM has identified alterations in the components of the PI3K pathway in 80% of primary GBMs, including amplification of receptor tyrosine kinases (RTKs), activating mutations in the PI3K catalytic subunit (PIK3CA), loss-of-function mutations in the PI3K regulatory subunit (PIK3R1), and loss of the major tumor suppressor of the pathway, phosphatase and tensin homologue (PTEN). Despite the universal upregulation of PI3K pathway signaling associated with these genetic alterations, clinical trials investigating PI3K inhibitors in GBM have failed due to insufficient antitumor activity and dose-limiting toxicities. To elucidate mechanisms of resistance to PI3K inhibition in GBM, we used a genome-wide functional CRISPR-Cas9 knockout screen to identify genes that mediate resistance to the dual PI3K/mTOR inhibitor XL765 in PTEN-null SF295 GBM cells. We identified reactivation of the PI3K pathway and inhibition of autophagy as potential resistance mechanisms to dual PI3K/mTOR inhibition. Using orthogonal approaches, we will functionally validate our candidate hits through mechanistic studies, with patient-derived models, and with in vivo orthotopic xenografts. Our findings will elucidate novel mechanisms of resistance to PI3K/mTOR inhibition in GBM and will streamline rational drug combinations aimed to overcome resistance, thus eventually maximizing therapeutic responses to PI3K inhibitors in GBM. Citation Format: Annalisa V. Ferrotta, Arianna Izawa-Ishiguro, Meaghan Grogan, Ingo K. Mellinghoff. A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5841.

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.

Phosphoinositide 3-Kinase (PI3K) is a central enzyme in a signaling pathway that mediates cellular responses to insulin and other growth factors. This enzyme phosphorylates the 3 position of phosphatidylinositol-4,5-bisphosphate to produce phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. A number of signaling proteins, including the Ser/Thr protein kinases, AKT and PDK1, contain pleckstrin homology domains that bind specifically to PIP3. Thus, the generation of PIP3 at the plasma membrane in response to activation of PI3K by growth factors results in the initiation of downstream Ser/Thr phosphorylation cascades that control a variety of cellular responses. The signaling pathway downstream of PI3K is highly conserved from worms and flies to humans and genetic analysis of the pathway has revealed a conserved role in regulating glucose metabolism and cell growth. Based on deletion of genes encoding the catalytic or regulatory subunits of PI3K in the mouse, PI3K mediates insulin dependent regulation of glucose metabolism, and defects in activation of this pathway result in insulin resistance. In contrast, mutational events that lead to hyperactivation of the PI3K pathway result in cancers. Activating mutations in PIK3CA, encoding the p110alpha catalytic subunit of PI3K or inactivating mutations in PTEN, a phosphoinositide 3-phosphatases that reverses the effects of PI3K, are among the most common events in solid tumors. PI3K driven tumors are FDG-PET positive and turning off PI 3-Kinase with PI3K inhibitors that are in human clinical trials results in an acute decline in FDG-PET signal that precedes tumor shrinkage. Importantly, there is increasing evidence that some tumors express high levels of insulin receptor and activate PI3K due to elevated serum insulin in patients with insulin resistance. These results suggest that elevations in serum insulin may partially explain the link between obesity, diabetes and cancers. The role of PI3K inhibitors for treating cancers in mouse models and in human trials will be discussed.

SummaryPhosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

Phosphoinositide 3-Kinase (PI3K) is a central enzyme in a signaling pathway that mediates cellular responses to growth factors. This enzyme phosphorylates the 3 position of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. A number of signaling proteins, including the Ser/Thr protein kinases, AKT and PDK1, contain pleckstrin homology domains that bind specifically to PIP3. Thus, the generation of PIP3 at the plasma membrane in response to activation of PI3K by growth factors results in the initiation of downstream Ser/Thr phosphorylation cascades that control a variety of cellular responses. The signaling pathway downstream of PI3K is highly conserved from worms and flies to humans and genetic analysis of the pathway has revealed a conserved role in regulating glucose metabolism and cell growth. Based on deletion of genes encoding the catalytic or regulatory subunits of PI3K in the mouse, PI3K mediates insulin dependent regulation of glucose metabolism, and defects in activation of this pathway result in insulin resistance. In contrast, mutational events that lead to hyperactivation of the PI3K pathway result in cancers. Activating mutations in PIK3CA, encoding the p110alpha catalytic subunit of PI3K or inactivating mutations in PTEN, a phosphoinositide 3-phosphatases that reverses the effects of PI3K, are among the most common events in solid tumors. We have generated mouse models in which a mutated form of the PIK3CA gene is expressed in a tissue specific and reversibly inducible manner. These mice develop cancers that are dependent on continuous expression of the mutant PIK3CA gene. The PIK3CA driven tumors are FDG-PET positive and turning off PI 3-Kinase with PI3K inhibitors that are in human clinical trials results in an acute decline in FDG-PET signal that precedes tumor shrinkage. These results suggest that the ability of PI3K to stimulate high rates of glucose uptake and metabolism may be critical for the survival of PIK3CA mutant tumors. The role of PI3K inhibitors for treating cancers in mouse models and in human trials will be discussed.

CN03-01: Mechanistic basis for treatment combinations with PI3K inhibitors.

Phosphoinositide 3-Kinase (PI3K) gamma is a lipid kinase that is regulated by G-protein-coupled receptors. It plays a crucial role in inflammatory and allergic processes. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits. The regulatory p101 subunit of PI3Kgamma binds to Gbetagamma and, thereby, recruits the catalytic p110gamma subunit to the plasma membrane. Despite its crucial role in the activation of PI3Kgamma, the structural organization of p101 is still largely elusive. Employing fluorescence resonance energy transfer measurements, coimmunoprecipitation and colocalization studies with p101 deletion mutants, we show here that distinct regions within the p101 primary structure are responsible for interaction with p110gamma and Gbetagamma. The p110gamma binding site is confined to the N terminus, whereas binding to Gbetagamma is mediated by a C-terminal domain of p101. These domains appear to be highly conserved among various species ranging from Xenopus to men. In addition to establishing a domain structure for p101, our results point to the existence of a previously unknown, p101-related regulatory subunit for PI3Kgamma.

Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Galpha q on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Galpha q-estrogen receptor hormone-binding domain fusion proteins (Galpha qQ209L-hbER or Galpha qQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but Galpha qQ209L-AA-hbER cannot activate the canonical Galpha q effector phospholipase Cbeta (PLCbeta). L-type Ca2+ current (I(Ca,L)) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Galpha q animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Galpha q does not require PLCbeta. To investigate the role of PI3K in this inhibitory effect, I(Ca,L) was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect I(Ca,L), but it fully restored I(Ca,L) density in both Galpha q transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110beta/p85alpha or p110gamma PI3K in the presence of PI(4,5)P2 also restored I(Ca,L) density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which sequesters PI(3,4,5)P3, reduced the peak I(Ca,L) density in wild-type myocytes by approximately 30%. Taken together, these results strongly suggest that the inhibitory effect of Galpha q on the cardiac LTCC is mediated by inhibition of PI3K.

Isoform‐selective phosphoinositide 3‐kinase inhibitors induce apoptosis in chronic lymphocytic leukaemia cells

The IKZF1 (Ikaros) gene encodes a DNA-binding protein that acts as a tumor suppressor in leukemia. Deletion of one Ikaros allele results in B-cell acute lymphoblastic leukemia (B-ALL) with a high rate of relapse and poor outcome. The mechanisms through which Ikaros suppresses leukemogenesis and that regulate Ikaros activity as a tumor suppressor in leukemia are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that control two pathways crucial for proliferation of leukemia cells: 1) cell cycle progression and 2) the phosphatidylinositol 3-kinase (PI3K) pathway. Using gain-of-function and loss-of-function experiments we demonstrate that Ikaros transcriptionally represses genes that promote cell cycle progression and the PI3K pathway and activates transcription of a gene that suppresses the PI3K pathway. In high-risk B-ALL with deletion of one Ikaros allele, we show that the function of Ikaros as a transcriptional regulator is impaired due to reduced binding at promoters of its target genes. Previous work shows that Ikaros DNA-binding affinity is regulated via direct phosphorylation by the pro-oncogenic kinase, CK2 (Casein Kinase II). We show that CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, further reducing Ikaros function. Treatment of primary high-risk B-ALL (with deletion of one IKZF1allele) using the CK2 specific inhibitor, CX-4945, restored Ikaros function as a transcriptional regulator of genes that control cell cycle progression and the PI3K pathway. Treatment with CK2 inhibitor was also associated with cell cycle arrest and reduced phosphorylation of the AKT kinase, a downstream PI3K pathway target. Using serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes, we demonstrated that Ikaros can repress transcription of its target genes through two distinct mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and the loss of H3K9ac; and 2) by an HDAC1-independent mechanism that is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac. The therapeutic efficacy of CK2 inhibition using CX-4945 against high-risk B-ALL was demonstrated in vivo using 4 different xenografts: 3 different high-risk primary pre-B-ALL patient-derived xenografts and Nalm6 xenografts. CX-4945 showed strong therapeutic effects in all 4 xenografts, as evidence by reduced leukemia cell numbers in bone marrow and in spleen, together with prolonged survival of treated xenograft animals. Expression analysis of Ikaros target genes in leukemia cells treated in vivo with CX-4945 revealed an expression pattern cell cycle regulatory and PI3K pathway genes that was highly similar to that observed with Ikaros overexpression. These data suggest that CK2 inhibition in vivo exerts its therapeutic effect on high-risk B-ALL by restoring Ikaros function as a transcriptional regulator of genes that promote cell cycle progression and the PI3K pathway. In summary, our results reveal that: 1) Ikaros functions as a tumor suppressor by suppressing cell cycle progression and the PI3K pathway; 2) Ikaros regulates transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator in vivo, and has a strong therapeutic effect in primary xenografts of high-risk B-ALL. These results provide support for the use of CK2 inhibitors in clinical trials for high-risk B-ALL. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. This abstract is also presented as Poster B10. Citation Format: Chunhua Song, Chandrika Gowda, XiaoKang Pan, Kimberly J. Payne, Sinisa Dovat. CK2 inhibition exerts a therapeutic effect in high-risk ALL by restoring IKZF1-mediated repression of cell cycle progression and the PI3K pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR09.

The phosphoinositide 3-kinase (PI3K) pathway signals not only for cell survival and proliferation in tumor cells but also controls DNA damage repair (DDR) and maintains HR (homologous recombination) steady-state the translational significance of which has been reported [Juvekar A. et al., 2012]. In breast cancers (BC), alterations of the PI3K pathway genes are both subtype-specific as well as contextual, and alterations of DDR pathway genes are one such contextual event occurring with the upregulation of the PI3K pathway. We hypothesized that there is contextual cooperation between the cellular signals of the PI3K and DDR pathways. We sought to identify the co-alterations of the PI3K and DDR pathway genes in our triple-negative BC (TNBC) patient cohort to (A) interrogate the contextuality of such alterations and (B) understand the mechanism of the contextual cooperation using an algorithmically chosen combination of pathway targeted drugs. We analyzed the somatic alterations profile of genes (FoundationOne, USA) in 264 BC patients (Avera Cancer Institute, SD, USA). We experimentally validated the cooperation between the pathways using alteration-guided targeted inhibitors; isoform-specific PI3K and PARP inhibitor(s) in the TNBC model, to demonstrate the efficacy of a combination of a node-specific targeted inhibitor(s) of the two pathways. The predominant type of mutation of genes in the PI3K pathway in our patients with TNBC was found in PTEN (Y68C, Y180*, loss, loss exons 1-5, and deletion exon1) and PIK3CA (amplification, E545K, H1047R). The other common mutation found was in TP53 (>80%) and somatic BRCA1/2 (~15%) genes. The interaction between the two pathways was evaluated, applying STRING10 to test the association at the highest 0.900 confidence views. The highest level of efficiency of drugs is demonstrated following the treatment of p110beta inhibitor (AZD 6482) in combination with PARP inhibitor (Olaparib/Talazoparib) plus DNA damaging agent (carboplatin) in BRCA-incompetent PTEN-null TNBC cells (SUM149) as compared to the efficiency of a combination of a PI3K pathway targeted inhibitor (of p110alpha) with a PARP inhibitor in BRCA-competent PTEN WT, but PIK3CA altered TNBC cells (BT20). The context-dependent synergy between the PI3K and DDR pathways in the TNBC model is comprehended at the levels of (1) load of DNA damage, (2) PARP activity, (3) of BRAC1/2 competency or HRD scores, and (4) mode of upregulation of PI3K pathway. We present an algorithm for a rational combination of the PI3K and the DDR pathway targeted drugs in TNBC. Citation Format: Nandini Dey, Jennifer Carlson Aske, Pradip De. Linking proliferative signal to DNA-damage signal in a tumor cell: A contextual synergy between the PI3K pathway and the DDR pathway in TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-02.