BI3352 Cancer: Cellular and Molecular Mechanisms and Therapeutics - Cancer Cell Signaling, Parts 1 & 2

Cell Signaling in Cancer Formation and Progression

Learning Objectives

• Understand the functional importance of cell signaling during tumor formation and progression.

• Comprehend how the PI3K pathway contributes to cancer growth.

• Appreciate the range of PI3K pathway inhibitors and their advantages/limitations.

• Recognize that different genetic drivers of the PI3K cascade show differential responses to PI3K pathway inhibitors.

Cell Signaling Basics

• Cell signaling involves:

  • Ligand: A chemical messenger.

  • Receptor: A protein that binds the ligand.

  • Response: The cellular outcome of the signaling event.

• Cell-intrinsic responses are triggered by exogenous stimuli, such as pathogens, acting on receptors.

• Cell-cell communication involves a source cell sending a signal to a target cell.

Importance of Cell Signaling in Cancer

• Cellular processes regulated by cell signaling:

  • Cell differentiation/transformation

  • Stem cell function

  • Cell growth and proliferation

  • Cell death (apoptosis)

  • Cell migration (invasion)

• Cell-extrinsic responses:

  • Angiogenesis

  • Immune response

  • ECM remodeling

• Genetic mutations in signaling pathways are crucial for cancer formation.

  • Germline mutations can predispose individuals to cancer.

Common Oncogenic Signaling Cascades

• 89% of cancers have at least one driver alteration in one of 10 mitogenic cancer pathways.

• 57% of patients have at least one actionable target.

• Three commonly deregulated signaling cascades:

  • RAS

  • PI3K

  • p53

The PI3K Pathway: Therapeutic Actionability

• Genomic profiling shows that a third of all solid cancers have activated PI3K signaling.

  • Prostate cancer: 32% of cases

  • Glioblastoma: 57% of cases

  • Breast cancer: 48-62% of cases

  • Colorectal cancer: 32-53% of cases

Function of the PI3K Pathway

• The PI3K pathway is activated by:

  • ATP

  • Amino acids

  • Growth factors

• Activation leads to signal transduction, resulting in:

  • Growth

  • Migration

  • Metabolism

  • Survival

  • Proliferation

  $NB: Act = AKT!!$

• Mutations in AKT occur in 3-5% of all cancers.

The PI3K Signaling Cascade

• Activation:

  • Cytokine, GPCR (G protein-coupled receptor), growth factor, and RTK (receptor tyrosine kinase) stimulate the pathway.

  • This leads to activation of PI3K, which converts $PIP2$ to $PIP3$.

  • PTEN reverses this conversion by dephosphorylating $PIP3$.

  • $PIP3$ recruits PDK1 and AKT to the plasma membrane.

  • AKT is then phosphorylated at threonine 308 (Thr308) by PDK1; mTORC2 phosphorylates AKT at serine 473 (Ser473).

• Downstream effects:

  • AKT phosphorylates other substrates like TSC1 and TSC2, influencing cell proliferation, survival, migration, and metabolism.

  • mTORC1, activated by Rheb, promotes ribosome biogenesis, protein translation, cell growth, and proliferation by phosphorylating 4EBP1 and S6K.

  • Apoptosis, cell cycle, and metabolism are also regulated in this cascade.

Genetic Drivers of the PI3K Pathway

• Oncogenes:

  • PIK3CA: Activating mutation/amplification.

    • Hotspot mutations in helical and kinase domains.

  • AKT1: Activating mutation/amplification (less common in AKT2 and AKT3).

    • Hot spot mutation in E17K – in PH domain rendering it constitutively associated with the PM, without the need for $PIP3$

• Tumor suppressors:

  • PTEN: Loss of function.

    • Mutation/truncation/epigenetic silencing/post-translational modifications.

Key Challenges in Targeting the PI3K Pathway

• Where to target:

  • Receptor level (which of the 58 RTKs to inhibit?).

  • Core intracellular components (PI3K, AKT?).

  • Downstream regulators (mTOR, eIF4E?).

• Which patients:

  • Do genetic variants within the PI3K pathway influence therapy responses?

  • Can we predict responders vs. non-responders?

  • Which disease stage is most efficacious?

• Safety:

  • Are there toxic/adverse side effects?

PI3K Inhibitors

(Diagram showing where PI3K inhibitors act within the PI3K pathway)

Class I PI3K Isoforms

• Class I phosphatidylinositol-3-kinase (PI3K) isoforms have distinct functions & expression profiles in tissues.

  (Table summarizing tissue expression, class I isoform, and GEMM KO effects for regulatory and catalytic subunits)

Types and Considerations for PI3K Inhibitors

• Inhibiting delta/gamma can influence the immune system (colitis & pneumonitis).

• Cytostatic agents (inhibit cancer cell growth but do not kill cancer cells).

• ATP-competitive or allosteric inhibitors.

• Pan-PI3K and isoform-specific PI3K inhibitors have oral bioavailability.

• Examples:

  • Pan-PI3K inhibitors: Buparlisib, Copanlisib, Sonolisib, Apitolisib

  • Isoform-specific PI3K inhibitors: Apelisib (BYL719), GSK2636771 (PI3Kb/d), AZD8186 (PI3Kb/d), Idelalisib (PI3Kd)

Pan-PI3K Inhibitors

• Buparlisib:

  • Effective against ER+ metastatic breast cancer (BELLE-2 trial).

    • Buparlisib + fulvestrant: median PFS = 6.9 months (n=576)

    • Placebo + fulvestrant: median PFS = 5 months (n=571)

  • Effective against PIK3CA mutant ER+ breast cancer:

    • PIK3CA mutant: Buparlisib + fulvestrant (median PFS = 7 months, n=87) vs. Placebo + fulvestrant (median PFS = 3.2 months, n=113)

    • PIK3CA wild-type: Buparlisib + fulvestrant (median PFS = 6.8 months, n=124) vs. Placebo + fulvestrant (median PFS = 6.8 months, n=126)

  • Associated with adverse side effects (BELLE-2 Phase III TRIAL).

  • Not FDA-approved for solid cancers (approved for lymphomas only).

FDA Approval

• Reviewed by the Centre for Drug Evaluation and Research (CDER) – USA.

• Approved: Benefits outweigh risks for the intended population.

• Not approved: Benefits do not outweigh risks.

PI3K-Isoform Specific Inhibitors

• Rationale:

  • Increased specificity.

  • Lower dose.

  • Reduce toxicity.

  • Immune cell specific.

• Targeting depends on the mutation type (e.g., PIK3CA, PTEN, PIK3CB, PIK3CD).

Precision Oncology

• Molecular profiling of tumors to identify actionable targets.

• Aim: tailoring targeted therapies for individual patients, based on their molecular and genetic characteristics.

• Examples: PIK3CA mutation = PI3Ka isoform specific inhibitor, whereas PIK3CB mutation = PI3Kb isoform specific inhibitor

PIK3CA Mutations

• Prevalent in cancer.

• Hotspots:

  • HELICAL DOMAIN: E545K, E542K

  • KINASE DOMAIN: H1047R

• Prevalent in multiple malignancies:

  • Endometrial cancer (>40% incidence).

  • Breast cancer (>35% incidence).

PI3Ka-Specific Inhibitor: Apelisib

• Effective against PIK3CA mutant ER+/HER2- advanced breast cancer.

  • PIK3CA mutations are present in 28%-46% of ER+/HER2− advanced breast cancer = poor prognosis (OS: 19.6 mo. vs 23.5 mo.).

  • Clinical benefit observed for all carriers with a PIK3CA mutation at known hotspot (E542X, E545X, and H1047X) detected by ctDNA.

    • PIK3CA mutant cohort: Median PFS improvement = 5.3 months (11 vs. 5.7 mo)

    • Non-PIK3CA mutant cohort: Median PFS improvement = 1.8 months (7.4 vs. 5.6 mo)

  • Challenge to administer: Around 1/3 patients showed grade 3 hyperglycemia.

• Apelisib (+ Fulvestrant) = FDA approved in May 2019 for metastatic ER+/HER2- breast cancer.

Preclinical Studies

• Show other PIK3CA mutant cancers respond to PI3Ka-isoform specific inhibitors.

  • PIK3CA mutations occur in approx. 4% of metastatic prostate cancer patients and correlates with poor outcome.

  • PIK3CA H1047R mutation causes invasive prostate carcinoma in mice, similarly to PTEN loss (albeit slower).

  • PIK3CA H1047R mutant prostate cancer is sensitive to PI3Ka inhibition (but not PI3Kb inhibition).

  • PTEN-deleted Pca is insensitive to PI3Ka inhibition alone.

• Pten-deleted PCa shares similar features with Pik3ca-activated PCa (Pik3caH1047R, Ptenfl/fl).

PTEN Loss and PI3K Inhibitor Sensitivity

• PTEN loss predicts for PI3Ka inhibitor resistance and pan-PI3K sensitivity.

• PDX model: PIK3CA mutant, PTEN-null lung metastasis from a breast cancer patient.

  • PI3Ka inhibitor unable to completely suppress the pathway, while pan-PI3K inhibitor is more effective.

PI3Ka-Inhibitor Resistance Mechanism

• PTEN inactivation.

• Apelisib treated ER+ mBrCa patient: characterization of 14 metastatic sites.

PI3Ka-Inhibitor Resistance in Advanced Breast Cancer

• Acquired PI3K pathway mutations drive PI3Ka inhibitor (apelisib or inavolisib) resistance in 50% of PIK3CA mutant patients (tissue or ctDNA analysis, n = 32 patients).

• Frequently acquired mutations include:

  • PTEN mutations = 18% cases

  • AKT1 mutations = 15% cases

  • PIK3CA mutations at a secondary site = 30% cases

Factors Limiting PI3K Inhibitor Response

• Methodologies:

  • Patient sample analysis

  • Genetic analysis

  • Disease stage

  • Dosing strategy – need for combination/consecutive therapies?

  • Biomarkers for response? E.g., certain genomic profiles. Multiple PIK3CA mutations increase sensitivity

• Key mechanisms for PI3Ki resistance:

  • Genomic variants (DNA mutations)

  • Pathway feedback loops – PI3K independent activation of AKT/mTOR

  • Transcriptional/epigenetic reprogramming – mediated by AKT downstream targets – FOXO3A, KMT2D

  • Metabolic switch – hyperinsulinemia

Overcoming Metabolic Mechanisms of PI3Ki Resistance and Reducing Toxicity

• Diet (ketogenic/fasting mimicking).

• Anti-hyperglycemics (SGLT2).

• Different dosing schedules and hyperglycemia monitoring.

• NEW DRUGS!

  • PI3K mutant selective inhibitors

    • Mutation specific (LOXO-783, H1047RMUT)

    • pan-PI3K mutation (RLY-2608, STX-478)

  • Covalent RAS-PI3K allosteric inhibitors: BBO-10203 (limited hyperglycemia?)

  (Diagram illustrating the metabolic effects in liver and pancreas and their effect on cancer growth and PI3K pathway reactivation)

• Mechanism of PI3Ki resistance due to metabolic changes:

  • Glycogen breakdown in liver increases glucose levels.

  • Cancer cells reduce glucose uptake through GLUT4

  • Pancreas increases insulin production as a result of high glucose levels.

  • Insulin activates the PI3K pathway through increased receptor tyrosine kinase leading to increased cancer growth

Summary

• The PI3K pathway is one of the most commonly activated pathways in human malignancies, presenting a valuable actionable target.

• Big pharma have developed a range of PI3K inhibitors (pan/isoform specific) but only one has been FDA approved (Apelisib, a PI3Ka-specific inhibitor) for ER+ metastatic breast cancer carrying PIK3CA/AKT/PTEN genetic aberrations.

• PI3K-isoform specific inhibitors offer a valuable opportunity for precision oncology.

• PI3K inhibitors are not easy to manage in the clinic – hyperglycaemia.

• PI3Ka-inhibitors are prone to resistance via the acquisition of additional mutations within the PI3K pathway – investigators are actively researching how to overcome this (new dose schedules, PI3K mutant specific inhibitors).

Precision Oncology: Key Challenges Faced

• Passenger vs Driver mutations

• Potential to miss actionable targets (e.g., mutation not previously identified)

• Over-interpretation of data (e.g., not all mutations are pathogenic)

• Resistance through the acquisition of new genetic variants

• Genomic studies are gene-centric, as opposed to pathway-centric

• Different study = unique approach to assessing genetic variants.

• Potential to miss actionable targets (new mutation/epigenetic)

• Absence of methodology standardisation

PI3K Pathway Activation

• PI3K activating mutation/amplification

• AKT activating mutation/amplification

• PTEN loss of function mutation

• Other modes of activation:

  • Mutations in other pathway components

  • PI3K pathway component post-translational modifications

  • Pathway cross-talk with RAS-MAPK and Wnt cascades

Targeting the PI3K/AKT/mTOR Pathway

Overview

• Illustrates where different inhibitors target the PI3K/AKT/mTOR pathway.

(Diagram of the PI3K/AKT/mTOR pathway indicating the targets of various inhibitors)

• PI3K Inhibitors

  • Pan-PI3K Inhibitors: Buparlisib (BKM120), Copanlisib (BAY 80-69460)

  • Isoform-specific PI3K Inhibitors: Apelisib (BYL719) (p110α), AZD8186 (p110β/δ), BAY1082439 (p110α/β/δ), GSK2636771 (p110β/δ)

• AKT Inhibitors (AKTi)

  • Afuresertib (GSK2110183), Capivasertib (AZD5363), Ipatasertib (GDC-0068), MK-2206, Uprosertib (GSK2141795)

• mTORC1/2 Inhibitors

  • Sapanisertib (INK-128, MLN0128) – weak PI3Ki

• Dual PI3K/mTORC1/2 Inhibitors

  • Apitolisib (GDC-0980), Dactolisib (BEZ235), Samotolisib (LY3023414) – also a DNA-PKi

• mTORC1 Inhibitors

  • Everolimus (RAD001), Sirolimus (Rapamycin), Temsirolimus (CCI-779)

AKT Signaling Details

• Inactive State:

  • AKT exists in an inactive conformation in the cytoplasm.

  • The PH domain binds to PI(4,5)P2

• Activation Process:

  • PI3K converts PI(4,5)P2 to PI(3,4,5)P3.

  • PTEN dephosphorylates PI(3,4,5)P3 back to PI(4,5)P2, inhibiting AKT activation.

  • PI(3,4,5)P3 recruits AKT to the plasma membrane via its PH domain.

  • PDK1 phosphorylates AKT at T308.

  • mTORC2 phosphorylates AKT at S473, fully activating it.

• Deactivation:

  • PP2A and PHLPP1 dephosphorylate AKT, leading to inactivation.

• Downstream Effects:

  • Active AKT phosphorylates various substrates including FOXO, PRAS40, GSK3, and TSC2.

  • These substrates regulate cell cycle, apoptosis, survival, proliferation, metabolism, and growth.

  • TSC2 inhibits Rheb, which activates mTORC1.

  • mTORC1 phosphorylates S6K and 4EBP1, promoting protein synthesis, inhibition of autophagy, and lipid synthesis.

Development of AKT Inhibitors

• Cytostatic agents

• Three main types:

  • AKT Allosteric Inhibitors: MK2206

  • AKT ATP-Competitive Inhibitors: Miransertib (ARQ-092), Ipatasertib, Capivasertib (FDA approved), GSK2141795

  • AKT Degraders: INY-03-041

FAKTION Trial

• Fulvestrant +/- AKT ATP-competitive inhibitor (Capivasertib) in metastatic ER+/HER2- breast cancer patients

• Median PFS improved by 5.5 months (10.3 mo vs. 4.8 months) in all patients (no genetic selection).

• No apparent increased effect in PIK3CA mutant/PTEN deficient cases.

• Hyperglycemia = 25% incidence (now exclude diabetics).

• FDA approved for PIK3CA/AKT/PTEN ER+/HER2- mBRCA.

Genomic Landscape of Prostate Cancer

(Bar graph showing the percentage of altered samples in prostate cancer across various genetic categories, including PI3K, DNA repair, epigenetic regulators, splicing, cell cycle, WNT-CTNNB1, and RAS-RAF-MAPK)

PI3K Pathway in Prostate Cancer

• Prostate cancer commonly harbors mutations in PI3K pathway components.

(Graph showing the frequency of gene alterations in PTEN, RRAGD, FOXO3, and other genes in primary and metastatic prostate cancer)

• 57% of primary cases and 75% of metastatic cases have alterations in the top 25 deregulated PI3K/AKT/mTOR pathway genes.

Clinical Trials in Prostate Cancer

• Clinical trials exploring PI3K/AKT/mTOR pathway-directed therapies in prostate cancer:

  • 79% Ineffective and/or toxic

  • 21% Active

  • Total = 42

AKT Inhibitors in Prostate Cancer

• AKT inhibitors are showing promise in the clinic against advanced prostate cancer (Crabb et al JCO 2020: PROCAID).

• CAPItello-280 - mCRPC (progression on ADT):

  • Docetaxel +/- Capivasertib

• CAPItello-281 - mHSPC-adeno (low PTEN) Phase III:

  • Abiraterone +/- Capivasertib

IPATential 150 Trial

• Metastatic castrate-resistant prostate cancer (mCPRC) patients with PTEN loss show increased radiographic progression free survival (rPFS): IPATential 150 trial

• IPATential 150 (Ipatisertib +/- abiraterone/Prednisone)

• Strong efficacy of AKT inhibitors associated with PTEN-deficient patients.

• BUT - currently discontinued….

AKT Inhibition in Breast Cancer

• AKT inhibition with catalytic inhibitor Ipatasertib is showing promise in breast cancer (LOTUS trial), but did not show efficacy in subsequent IPATunity Trial

• Tumor type (not genetic alteration) is important for AKT inhibitors in the context of paclitaxel: advanced TNBC is sensitive to Ipatasertib combined with paclitaxel, while advanced ER+/HER2 breast cancer is not.

• LOTUS metastatic TNBC trial:

  • Paclitaxel +/- AKTi (Ipatisertib)

• IPATunity130 HR+ HER2- metastatic breast cancer trial:

  • Paclitaxel +/- AKTi (Ipatisertib) – PIK3CAMUT/AKT1/PTEN altered

AKT-Inhibitor Resistance

• Multiple mechanisms:

  • AKT kinase independent functions

  • PI3K/AKT/mTOR pathway feedback loops activate AKT downstream targets

  • Incomplete suppression of the pathway

• Other considerations:

  • Impact of genomic landscape on treatment response – Can PI3K pathway mutations predict AKT inhibitor response?

  • Does the tumour type/stage influence AKT inhibitor sensitivity?

  • Role of the tumour microenvironment?

AKT Kinase-Independent Functions

• ATP-competitive inhibitors only target the catalytic domain, but AKT can still perform non-kinase functions.

• AKT can still play a role as a protein scaffold.

• AKT can still compete and bind to its substrates.

• AKT can still undertake specific functions that require an open conformation.

• AKT can still move to different subcellular locations (e.g., nucleus).

AKT Degraders

• AKT degraders: INY-03-041

• More durable AKT signalling suppression than GDC-0068 (Ipatasertib) ATP competitive AKT inhibitor

• PROTAC = AKT binding + LINKER + E3 ligase

• Advantage: Eradicate AKT independent functions

• Potential limitations: Unknown impact on side effects

Incomplete Suppression of AKT

(Diagram illustrating AKT inhibition and showing alternate pathways for cell survival/growth, cell cycle progression, and metabolism)

Combination Therapies

• AKT inhibitor + Chemotherapy (Paclitaxel – breast cancer, Docetaxel – prostate cancer)

• AKT inhibitor + RAS/MAPK inhibitor

• AKT inhibitor + mTOR inhibitors

• AKT inhibitor + Hormone therapy (Breast/Prostate)

• AKT inhibitor + SGK inhibitor

SGK Up-Regulation

• A potential AKT inhibitor resistance mechanism

  (diagram showing SGK1 IHC in prostate cancer)

• AKTi resistance = high SGK1

AKT and SGK Co-inhibition

(Diagram showing structural similarities between AKT and SGK)

• AKT and SGK are structurally similar:

  • Shared domain structure

  • Similar phosphorylation sites in the catalytic and c-terminal domains

  • Shared (and unique) substrates

Efficacy of AKT+SGK Co-inhibition

• AKT+SGK co-inhibition is more efficacious than monotherapy in a preclinical breast cancer xenograft model

• BT474 ER+HER2+ breast cancer xenograft model

• MK2206 = allosteric AKT inhibitor

• 14h = SGK inhibitor

Targeting mTOR

(Diagram showing where mTOR inhibitors act in the PI3K/AKT/mTOR pathway)

mTOR as an Actionable Target

• Mechanistic target of rapamycin (mTOR) is a dual-specificity protein kinase.

• mTOR is assembled into protein complexes known as mTORC1, mTORC2, mTORC3 and mTORC4.

• Key functions: regulating cell growth, survival, metabolism, immune function and drug resistance.

• mTOR mutations are rare in cancer

mTOR Inhibitors

• 1st generation: Rapalogs – allosteric inhibitors (Rapamycin/evirolimus/temsirolimus)

• 2nd generation ATP kinase domain mTORC1/2 inhibitors (AZD8055)

• 3rd generation = Rapalinks

• 1st mTOR inhibitors FDA approved

• No mTORC2 inhibitors have been approved for clinical use.

• mTORC1i can benefit some patients

Targeting mTOR Signaling

• Considerations:

  • AKT signalling remains active

  • mTOR complex functional redundancy (dual mTORC1/2 inhibitors?)

  • Are PI3K pathway mutations (or others) predictive of sensitivity?

• Everolimus:

  • PIK3CA mutations = not predictive

  • TSC1/2 mutations = predictive

Clinical Trials of mTORC1/2 Inhibitors

• mTORC1/2 inhibitor Vistusertib:

  • OCTOPUS trial – ovarian high-grade serous carcinoma = no overall benefit (with or without paclitaxel)

  • VICTORIA trial – ER+ advanced endometrial cancer = showing promise with anastrozole (Progression free survival = 5.2 months vs 1.9 months with anastrozole alone)

• mTORC1/2 inhibitor Sapanisertib (ATP-competitive):

  • ECOG-ACRIN EA2161- advanced pancreatic neuroendocrine tumours that are rapalog-resistant = no benefit (+ hyperglycemia prevalent)

• mTORC1/2 inhibitor Onatasertib (CC-223, ATP-competitive):

  • Chemotherapy resistant non-pancreatic neuroendocrine tumours (with somatostatin) = showing promise + manageable safety profile, 83% had stable disease, 7% had partial response

mTOR Inhibitor Limitations

• Toxicity

  • Hyperglycemia toxicity, stomatitis, diarrhea, rash and pneumonitis – improved management needed (e.g., Dexamethasone mouthwash decreases everolimus-induced stomatitis)

• Resistance mechanisms

  • Incomplete suppression of the PI3K/AKT/mTOR pathway - increased RAS/MAPK signalling via alleviation of S6K inhibition of IRS1

  • mTOR mutations– prevent ATP-competitive inhibitors from binding

  • mTORC1/2/3/4 redundancy (mTORC3 is resistant to mTORC1 inhibitors)

  • Sustained eIF4E activity (brings ribosomes to mRNA for protein synthesis) – combine mTOR targeted therapies with eIF4E inhibitors and/or mRNA translation blockade.

  • Metabolic rewiring

• Lack of predictive biomarkers (rare mTOR mutations)

Mechanisms of mTOR Inhibitor Resistance

(Diagram illustrating mechanisms of mTOR inhibitor resistance)

The Future for PI3K-Directed Therapies

• Combination therapies are likely to be the way forward in improving efficacy.

  • Optimal dosing strategies (intermittent, parallel, consecutive?).

  • Informed patient stratification strategies.

• Better clinical management of patients to reduce side effects (e.g., no diabetic patients on AKT inhibitors, diet, anti-hyperglycemics).

• Precision oncology advances:

  • Which molecular features predict response to which PI3K pathway targeted therapy?

  • Do PI3K-mutant specific inhibitors show efficacy in the clinic?

• Development of new drugs – researchers are beginning to explore going even further downstream (e.g., eIF4E inhibitors downstream of mTORC1/4), or dual PI3K/mTOR inhibitors (Gedatolisib).