Functional characterisation of a novel class of in-frame insertion variants of KRAS and HRAS

Abstract

Mutations in the RAS genes are identified in a variety of clinical settings, ranging from somatic mutations in oncology to germline mutations in developmental disorders, also known as ‘RASopathies’, and vascular malformations/overgrowth syndromes. Generally single amino acid substitutions are identified, that result in an increase of the GTP bound fraction of the RAS proteins causing constitutive signalling. Here, a series of 7 in-frame insertions and duplications in HRAS (n = 5) and KRAS (n = 2) is presented, resulting in the insertion of 7–10 amino acids residues in the switch II region. These variants were identified in routine diagnostic screening of 299 samples for somatic mutations in vascular malformations/overgrowth syndromes (n = 6) and in germline analyses for RASopathies (n = 1). Biophysical characterization shows the inability of Guanine Nucleotide Exchange Factors to induce GTP loading and reduced intrinsic and GAP-stimulated GTP hydrolysis. As a consequence of these opposing effects, increased RAS signalling is detected in a cellular model system. Therefore these in-frame insertions represent a new class of weakly activating clinically relevant RAS variants.

RASInternal Tandem Duplication Disrupts GAP-binding to Activate Oncogenic Signaling

“…However, because information on the specific missense variant identified duplications in their study of metastatic colorectal cancer, but did not further elaborate on the clinical characteristics of these cases (9). As compared to the reports from White et al (11) and Eijkelenboom et al (12), our data suggest more robust downstream activation of MAPK by the 10 amino acid duplication in both KRAS and NRAS. We also provide data gathered from in-depth in vitro evaluation of ITD activity, and additionally describe the first crystal structure of the RAS ITD.…”

“…ITDs in general are indeed rare in somatic malignancies, but functional and biochemical data are consistent with an oncogenic phenotype (11). Eijkelenboom et al have reported that RAS ITDs (particularly involving HRAS) are slightly more common in vascular malformation/overgrowth syndromes (2% prevalence) (12). As the oncogenic and other biologic properties of RAS isoforms (KRAS vs. NRAS vs. HRAS) differ, we likewise postulate that the clinical effects of their respective ITDs may vary as well between RAS family members.…”

“…The prevalence of such RAS ITDs in CRC is unknown, and at the time of initial diagnosis (2015), we found literature reports of only three prior CRC patients with this type of duplication in KRAS (2,9,10). Subsequently, other reports of RAS ITDs have emerged including one report of a pediatric patient with a hematologic malignancy (11) and a report of RAS-family ITDs in up to 2% of vascular malformation/overgrowth syndrome cases (12).…”

RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling

“…A prior report of a 7-amino acid RAS ITD identified in a pediatric hematologic malignancy provided functional and biochemical data supporting an oncogenic phenotype (6). Eijkelenboom et al (7) have reported that RAS ITDs (particularly involving HRAS, ranging in size from 7 to 10 amino acids) are slightly more common in vascular malformation/overgrowth syndromes (2% prevalence). The different biological properties of RAS isoforms (KRAS, HRAS, and NRAS) are mainly dictated by differences in the hypervariable region (i.e.…”

“…The prevalence of such RAS ITDs in CRC is unclear, and at the time of initial diagnosis (2015), we found literature reports of only three prior CRC patients with this type of duplication in KRAS (3)(4)(5). Subsequently, other reports of RAS ITDs have emerged, including one report of a pediatric patient with a hematologic malignancy (6), and a report of RAS-family ITDs in up to 2% of vascular malformation/overgrowth syndrome cases (7). We sought to elucidate the crystal structure of this previously uncharacterized NRAS ITD, and performed extensive functional characterization to determine its effects on RAS biological activity in relation to other known oncogenic RAS mutations.…”