Paper - Review

DOI: 10.1016/j.cell.2020.09.044

Summary

KRAS mutation
→ are the most common genetic alterations
← in 1⃣ lung 2⃣ colorectal 3⃣ pancreatic cancers

Direct inhibition (← of KRAS onco-protines)
→ has been a long-standing pursuit
← in precision oncology

Medicinal chemistry
→ have established → inhibitors targeting KRAS(G12C)
← in 13% of lung adeno-carcinomas

1⃣ Their discovery 2⃣ Their mechanism ← of actions
← coupled with emerging clinical data ← from patients treated with these drugs
→ have sparked a renewed enthusiasm ← in 1⃣ the study of KRAS 2⃣ the study of its therapeutic potential

❓: how these advances are reshaping
→ 1⃣ the fundamental aspects of KRAS oncoprotein biology
→ 2⃣ the strides being made → toward improving patient outcomes

Introduction

1⃣ KRAS 2⃣ the highly related NRAS
→ GTP → to GDP

They
→ control diverse cellular functions
← by cycling ← between 1⃣ an active, GTP-bound 2⃣ an inactive, GDP-bound conformation

GTPase activity (← of KRAS)
→ is enhanced
← by GTPase-activating proteins (GAPs)
← e.g. NFI

Exchange of GDP → for GTP
→ is enhanced
← by GEFs ← guanine-nucleotide exchange factors
← e.g. SOS1/2

KRAS
→ activate several effector proteins
→ to control diverse cellular functions

The best-studied effectors include
← 1⃣ RAF kinases 2⃣ PI3K ← the catalytic subunit of (phosphatidylinositol 3-kinase ← PI3K)

KRAS-GTP
→ binding to RAF kinase
→ stimulated their (dimerization & activation)
→ triggers stepwise activation ← of 1⃣ progression 2⃣ proliferation
← a pathway ← that drives cell cycle 1⃣ progression 2⃣ proliferation

Effective targeting (← of KRAS signaling)
→ to achieve in patients

Competitive inhibition (← of nucleotide binding)
→ has not ❌ been feasible
→ for RAS GTPases
∵ their high affinity → for the nucleotide

Blocking localization of KRAS
← at the plasma membrane
← a key component for its activation
→ as been ineffective 👎
∵ Multiple compensatory pathways → regulate this process

Targeting effector signaling
← downstream of KRAS
→ has not ❌ yielded significant clinical benefits
∵ Paradoxical signaling activation ← triggered by the inhibitor
∵ On-target toxicity ← limiting the maximum tolerated dose

❗: review
→ 1⃣ the evolution of these exciting new therapies 2⃣ their mechanisms of action 3⃣ cellular effects in pre-clinical studies

KRAS activation in cancer

KRAS mutations
→ are found predominantly
← in 1⃣ lung 2⃣ pancreatic 3⃣ colorectal

Mutation
← in 1⃣ NRAS 2⃣ HRAS
→ are also found ← in cancer patients
❕: at a low frequency than KRAS

KRAS mutations
→ result in single (amino acid substitutions)
← which activate the onco-protein
← by hindering its ability to hydrolyze GTP

The distribution (← of KRAS mutation alleles)
→ differs across tumors
← G12C comprising 50% of KRAS mutations ← in lung cancer
← G12D being the most common allele ← in 1⃣ pancreatic 2⃣ colorectal cancer

The KRAS (G12C) allele
→ is found → at a lower frequency
← in 1⃣ colorectal cancer 2⃣ uterine cancer 3⃣ mesothelioma 4⃣ pancreatic cancer

RAS onco-proteins
→ have measurable (intrinsic GTP hydrolysis rates)
← in biochemical assays
∵ 1⃣ Inconsequential → for cellular function 2⃣ KRAS onco-proteins were constitutively active

Allele-specific inhibitors targeting a mutated cysteine residue

Direct (KRAS onco-protein inhibition)
→ has been a long-standing objective
← in precision medicine

The discovery of inhibitors
← that selectively target KRAS(G12C)
→ was a ground-breaking advance in this quest

The inhibitors
→ bind covalently → to the mutant cysteine residue
→ occupy a pocket ← in the switch II region (SIIP)
← when KRAS(G12C) is ← in its inactive GDP-bound state

Structure-based optimization
→ led to inhibitors
←with progressively higher affinities → for KRAS(G12C)

These suppress
→ 1⃣ KRAS(G12C) activation 2⃣ attenuate proliferation 3⃣ induce cell death
→ to varying degrees across (tumor models)

ARS1620
→ inhibits 👎 → (tumor growth)
← in xenograft models
→ consolidating inactive state-selecting inhibition

The structure-activity relationships
← of the ARS compounds
→ furthered → the development of (even more potent G12Ci)

These inhibitors
→ have distinct chemical structures
→ although, they share (an acrylamide warhead)
← which engages G12C

Two of these
← 1⃣ sotorasib 2⃣ MRTX849
→ have half-maximal growth inhibitory concentration (IC50) values ← in the low nano-molar range
→ potently suppress xenograft tumor growth ← in mice

Their enhanced potency
→ is reportedly
∵ An enhanced interactions ← with the H95 residue
← in the α3 helix of KRAS(G12C)

❗: (Sotorasib & MRTX849) show
→ clinical activity
← in patients with KRAS (G12C) mutant tumors

Mechanism of inactive state-selective KRAS(G12C) inhibition

❓: how do inactive (state-selective drugs) inhibit KRAS(G12C) ?
← an onco-protein conventionally thought to be constitutively active

KRAS(G12C)
→ undergoes nucleotide cycling ← in cancer cells
→ fluctuates ← between (its active 🆚 inactive states)

Secondary mutations
← which completely block hydrolysis
← e.g. 1⃣ A59G 2⃣ Q61L
→ increase → baseline KRAS(G12C) activation
→ attenuate → the response to G12Ci treatment

Intact GTPase activity
→ is required → for KRAS(G12C) inhibition
← by inactive state-selective drugs

Drug ← binding to KRAS(G12C)
→ prevents → its re-activation
← by 1⃣ nucleotide exchange 2⃣ traps the onco-protein
← in the inactive state

❓: how KRAS(G12C)
→ undergoes → sufficient hydrolysis
→ to enable inhibition
← by inactive state-selective drugs

❗: KRAS(G12C) → is unique
← among commonly occurring KRAS mutants
← in that it has (a high intrinsic hydrolysis rate)

Initial clinical effects

Indicative ( ←of the tremendous progress)
→ made in the 7 years ← since their discovery
← several (inactive state-selective KRAS(G12C) inhibitors) → have entered clinical trilas

Clinical data → suggest that
→ a wide therapeutic index → for inhibitors
← e.g. 1⃣ sotorasib 2⃣ MRTX849

❗: Un-anticipated toxicity ← in 1⃣ animal 2⃣ early-phase clinical studies
→ has halted the clinical development (← of other compounds)

All available G12Ci
→ have a selectivity threshold
← in pre-clinical studies

The covalent nature (← of drug binding)
→ may lead → to off-target effects
← caused by non-selective interaction
← with cysteine residues ← in other proteins

Global proteomics analysis
← aiming → to identify all cysteine residues
← which are covalently modified by the drug
→ show clear selectivity for KRAS(G12C)
← in cells harboring this mutation

Efficacy data → are emerging
← from the clinical trial investigating sotorasib

Nearly half ← in lung cancer
→ had at least a partial response
→ at an initial radiologic evaluation
∴ Early adaptation → limits → the effect of therapy

The majority ← of (colorectal cancer patients)
→ did NOT ❌ have → a radiologic response
→ to treatment

1⃣ Sotorasib 2⃣ MRTX849
→ differ
← in 1⃣ their elimination half-lives 2⃣ administration schmems

Lack of a radiologic treatment
→ response ← in most patients
←with colorectal cancer
∴ Not ❌ (all KRAS mutant cancer) → are dependent
← on this onco-protein for growth

BRAF(V600) mutant ← colorectal cancers
→ are insensitive → to RAFi treatment
∵ 1⃣ higher activation of EGFR 2⃣ formation of RAFi-insensitive RAF dimers

Cancers
→ are independent (← of KRAS(G12C))
∴ Requires evidence of (1⃣ potent 2⃣ sustained target inhibition)
← without an effect on phenotypes
← which associated with a selective growth advantage

Adaptation to treatment

G12Ci treatment
→ only briefly suppress
→ KRAS signaling ← in cells

Initial onco-protein signaling inhibition
→ is accompanied
← by 1⃣ re-accumulation of active KRAS 2⃣ re-activation of ERK signaling

This pattern
→ is consistent with adaptation
← a process which is well described ← in response to 1⃣ RAF 2⃣ MEK inhibitors

❗: Accumulation of (active KRAS)
Compensatory activation (← of 1⃣ receptor tyrosine kinases (RTKs) 2⃣ SOS1/2)
← which are both feedback suppressed by ERK
→ is in (large part responsible)
→ for the adaptive changes noted ← during G12Ci treatment

RTKs
→ may modulate adaptation to G12Ci treatment
← in two ways

RTK activation
→ shifts KRAS(G12C)
→ to its GTP-bound conformation
← which is insensitive to the drug

RTKs
→ can also bypass inhibition
← in a G12C-independent manner

The role of PI3K/MTOR
→ is supported
← by the presence of anti-proliferative synergy
← when the G12Ci is combined ← with 1⃣ PI3K 2⃣ MTOR inhibitors

❗: Wild-type RAS proteins
→ are feedback activated
← following G12Ci treatment
❓: Whether 1⃣ this is compensatory response? 2⃣ one that drives adaption?

∴ Further testing is needed
→ to show that → wild-type RAS down-regulation enhances G12C inhibition
→ to determine → the relative contribution of 1⃣ NRAS 2⃣ HRAS 3⃣ wild-type KRAS

Adaptive signaling
→ have been integrated with single-cell modeling
→ to show that adaptation to G12Ci treatment occurs rapidly
← in a non-uniform manner across cancer cell populations

The divergent response
→ is modulated
← by productio of new KRAS(G12C)
← by its distribution between (the active/drug-insensitive 🆚 inactive/drug-sensitive) states

New KRAS(G12C)
→ is synthesized
← in response to suppressed ERK signaling
New KRAS(G12C)
→ is more susceptible → to activation
← by nucleotide exchange than baseline KRAS(G12C)

❗: Why G12Ci re-challenge
→ is less effective
← than initial treatment
❗: How active KRAS
→ can re-accumulate ←during G12Ci treatment
← even though the drug binds ← in a covalent/irreversible manner

Acquired resistance

Acquired resistance
→ limits 1⃣ the clinical benefit 2⃣ the effect of G12Ci treatment

❗: How acquired resistance
→ to G12Ci treatment might emerge

Predicted → to attenuate target inhibition
← in three manners:
1⃣ secondary mutations on KRAS(G12C)
← which 1⃣ impede (drug binding) 2⃣ enhance the propensity
2⃣ genetic events
← which consolidate the up-regulation of KRAS
3⃣ activation of nucleotide exchange
← through 1⃣ alteration in GEFs 2⃣ other up-stream intermediates

Predicted → to mediate resistance
← while the target remains inhibited ← include two manners
1⃣ activation of down-stream signaling
← through 1⃣ gain-of-function events ← in (RAF & MEK & ERK) 2⃣ loss-of-function events ← in RB1 3⃣ cyclin-dependent kinase inhibitors
2⃣ activation of (parallel signaling pathways)
← through 1⃣ loss of (NF1 & PTEN) 2⃣ activating mutations ← in other RAS GTPases

Maximizing therapeutic effects

Clinical active KRAS(G12C) inhibitors
→ have a tremendous potential
→ to affect patient care

Optimizing monotherapy

Drug exposure
→ directly correlates
← with 1⃣ the magnitude of ERK inhibition 2⃣ response in patients ← with BRAF(V600) mutant tumors treated with a RAFi

∴ Efforts
← to establish inhibitors ← with an even higher affinity ← for the inactive state of KRAS(G12C)
→ will ensure → more (potent & durable inhibition)
← by delaying adaptation

1⃣ alternative formulations 2⃣ administration schemes
← enabling (high drug exposure) ← in patients
→ may enhance → 1⃣ the response rate 2⃣ its magnitude

❗❓: whether sotorasib → produces (stronger anti-tumor effects)
← when administered to patients ← on a twice daily schedule

1⃣ Intermittent 2⃣ pulsatile therapy
→ has been suggested
→ to prolong the response
→ 1⃣ to inhibition 👎 of (1⃣ BCR-ABL 2⃣ EGFR 3⃣ BRAF) 2⃣ to enable multi-modal pathway inhibition

1⃣ Intermittent 2⃣ pulsatile therapy
→ improve anti-tumor immunity

❓: the duration of (target engagement) ← in washout studies
❓: whether intermittent dosing mitigate → the adaptive changes

Pharmacodynamic endpoints
→ must be implemented ← in clinical assessment
← of the G12Ci treatment response

Rational drug combinations

G12Ci
→ are well suited → for combination therapy
∵ A wide therapeutic index

Co-targeting upstream signaling

Suppressing (nucleotide exchange)
→ increases residency of KRAS(G12C)
← in its inactive/drug-sensitive state

Suppressing (nucleotide exchange)
→ enhances → the therapeutic effect ← of G12Ci

∵ RTK activation → is a key stimulus → for exchange

RTK inhibitors directly increase
→ the ability of the G12Ci
→ to engage its target

EGFR
→ has emerged ← as an early lead

G12Ci combinations
← with 1⃣ cetuximab 2⃣ erlotinib
→ are entering clinical testing in patients
← with 1⃣ colorectal cancer 2⃣ lung cancer

Direct suppression ← of nucleotide exchange downstream
← of multiple RTKs
→ is also effective
← by using SOS1-specific small interfering RNAs (siRNAs)

SHP2i
→ enhance → 1⃣ the anti-proliferative 2⃣ anti-tumor effects ← of G12Ci

Co-targeting parallel signaling

The effect ← on (1⃣ PI3K 2⃣ AKR 3⃣ MTOR) signalin
→ is more subtle
∵ Activation of PI3K
→ requires input ← from 1⃣ RAS 2⃣ RTKs
∵ Both signals ← may need
→ to be inhibited → for complete AKT blockade

Models
← where pERK inhibition is coupled ← with suppressed pS6K/pS6
→ exhibit a more pronounced anti-proliferative effect

Combined inhibition
← of (1⃣ PI3K 2⃣ MTOR) alongside KRAS(G12C)
→ has more pronounced anti-tumor effects
← than either drug alone

Co-targeting downstream signaling

An intuitive approach
→ to enhance KRAS(G12C) inhibition
→ is ← by targeting 1⃣ RAF dimers 2⃣ MEK 3⃣ ERK

❗: Inhibition of ERK output
→ releases → the feedback suppression of SOS1
→ which may impede → (target engagement) ← by the drug

MEKi treatment
→ does NOT ❌ appear
→ to significantly enhance → the effect of 1⃣ ARS1620 2⃣ MRTX849
MEKi treatment
→ has been reported → to enhance the anti-tumor effect of sotorasib

Co-targeting cell cycle checkpoints

KRAS signaling
→ is activation of the CyclinD:CDK4/6
→ leading to RB1 hyper-phosphorylation & progression
← through the G1/S cell cycle checkpoint

Co-targeting immune checkpoints

KRAS signaling
→ has immuno-suppressive effect
← on the tumor micro-environment

∴ KRAS(G12C) inhibition
→ might enhance → immune check-point inhibition

Sotorasib
→ enhanced expression of (inflammatory chemokines)
→ leading to enhanced (T-cell infiltration)

❓: how KRAS(G12C)
→ leads to immuno-suppression

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