KRAS insertion mutations are oncogenic and exhibit distinct functional properties.

Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-Ras(G60_A66dup) and K-Ras(E62_A66dup) proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications

Structural and functional analyses of a germline KRAS T50I mutation provide insights into Raf activation.

A T50I substitution in the K-Ras interswitch domain causes Noonan syndrome and emerged as a third-site mutation that restored the in vivo transforming activity and constitutive MAPK pathway activation by an attenuated KrasG12D,E37G oncogene in a mouse leukemia model. Biochemical and crystallographic data suggested that K-RasT50I increases MAPK signal output through a non-GTPase mechanism, potentially by promoting asymmetric Ras:Ras interactions between T50 and E162. We generated a switchable system in which K-Ras mutant proteins expressed at physiologic levels supplant the fms like tyrosine kinase 3 (FLT3) dependency of MOLM-13 leukemia cells lacking endogenous KRAS and used this system to interrogate single or compound G12D, T50I, D154Q, and E162L mutations. These studies support a key role for the asymmetric lateral assembly of K-Ras in a plasma membrane-distal orientation that promotes the formation of active Ras:Raf complexes in a membrane-proximal conformation. Disease-causing mutations such as T50I are a valuable starting point for illuminating normal Ras function, elucidating mechanisms of disease, and identifying potential therapeutic opportunities for Rasopathy disorders and cancer

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 “Gatekeeper” F691L Mutation with PLX3397

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKIs) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase “gatekeeper” residues, which control access to an allosteric pocket adjacent to the ATP-binding site, have been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first co-crystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved DFG motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 “Gatekeeper” F691L Mutation with PLX3397

UnlabelledTyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.SignificanceWe report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop

Association of Combination of Conformation-Specific KIT Inhibitors With Clinical Benefit inPatients WithRefractory Gastrointestinal Stromal Tumors A Phase 1b/2a Nonrandomized Clinical Trial

IMPORTANCE Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), are driven by activating mutations in tyrosine kinases and may initially respond to kinase inhibitors but frequently relapse owing to outgrowth of heterogeneous subclones with resistance mutations. KIT inhibitors commonly used to treat GIST (eg, imatinib and sunitinib) are inactive-state (type II) inhibitors. OBJECTIVE To assess whether combining a type II KIT inhibitor with a conformationcomplementary, active-state (type I) KIT inhibitor is associated with broad mutation coverage and global disease control. DESIGN, SETTING, AND PARTICIPANTS A highly selective type I inhibitor of KIT, PLX9486, was tested in a 2-part phase 1b/2a trial. Part 1 (dose escalation) evaluated PLX9486 monotherapy in patients with solid tumors. Part 2e (extension) evaluated PLX9486-sunitinib combination in patients with GIST. Patients were enrolled from March 2015 through February 2019; data analysis was performed from May 2020 through July 2020. INTERVENTIONS Participants received 250, 350, 500, and 1000mg of PLX9486 alone (part 1) or 500 and 1000mg of PLX9486 together with 25 or 37.5mg of sunitinib (part 2e) continuously in 28-day dosing cycles until disease progression, treatment discontinuation, or withdrawal. MAIN OUTCOMES AND MEASURES Pharmacokinetics, safety, and tumor responseswere assessed. Clinical efficacy end points (progression-free survival and clinical benefit rate) were supplemented with longitudinal monitoring of KIT mutations in circulating tumor DNA. RESULTS A total of 39 PLX9486-naive patients (median age, 57 years [range, 39-79 years]; 22 men [56.4%]; 35 [89.7%] with refractory GIST) were enrolled in the dose escalation and extension parts. The recommended phase 2 dose of PLX9486 was 1000mg daily. At this dose, PLX9486 could be safely combined with 25 or 37.5mg daily of sunitinib continuously. Patients with GIST who received PLX9486 at a dose of 500mg or less, at the recommended phase 2 dose, and with sunitinib had median (95% CI) progression-free survivals of 1.74 (1.54-1.84), 5.75 (0.99-11.0), and 12.1 (1.34-NA) months and clinical benefit rates (95% CI) of 14%(0%-58%), 50% (21%-79%), and 80% (52%-96%), respectively. CONCLUSIONS AND RELEVANCE In this phase 1b/2a nonrandomized clinical trial, type I and type II KIT inhibitors PLX9486 and sunitinib were safely coadministered at the recommended dose of both single agents in patients with refractory GIST. Results suggest that cotargeting 2 complementary conformational states of the same kinase was associated with clinical benefit with an acceptable safety profile