Diverse alterations associated with resistance to KRAS(G12C) inhibition.

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.

The subventricular zone is able to respond to a demyelinating lesion after localized radiation.

Radiation is a common tool in the treatment of brain tumors that induces neurological deficits as a side effect. Some of these deficits appear to be related to the impact of radiation on the neurogenic niches, producing a drastic decrease in the proliferative capacity of these regions. In the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles is the main neurogenic niche. Neural stem/precursor cells (NSCs) within the SVZ play an important role in brain repair following injuries. However, the irradiated NSCs' ability to respond to damage has not been previously elucidated. In this study, we evaluated the effects of localized radiation on the SVZ ability to respond to a lysolecithin-induced demyelination of the striatum. We demonstrated that the proliferation rate of the irradiated SVZ was increased after brain damage and that residual NSCs were reactivated. The irradiated SVZ had an expansion of doublecortin positive cells that appeared to migrate from the lateral ventricles toward the demyelinated striatum, where newly generated oligodendrocytes were found. In addition, in the absence of demyelinating damage, remaining cells in the irradiated SVZ appeared to repopulate the neurogenic niche a year post-radiation. These findings support the hypothesis that NSCs are radioresistant and can respond to a brain injury, recovering the neurogenic niche. A more complete understanding of the effects that localized radiation has on the SVZ may lead to improvement of the current protocols used in the radiotherapy of cancer.

Subventricular zone localized irradiation affects the generation of proliferating neural precursor cells and the migration of neuroblasts.

Radiation therapy is a part of the standard treatment for brain tumor patients, often resulting in irreversible neuropsychological deficits. These deficits may be due to permanent damage to the neural stem cell (NSC) niche, damage to local neural progenitors, or neurotoxicity. Using a computed tomography-guided localized radiation technique, we studied the effects of radiation on NSC proliferation and neuroblast migration in the mouse brain. Localized irradiation of the subventricular zone (SVZ) eliminated the proliferating neural precursor cells and migrating neuroblasts. After irradiation, type B cells in the SVZ lacked the ability to generate migrating neuroblasts. Neuroblasts from the unirradiated posterior SVZ did not follow their normal migratory path through the irradiated anterior SVZ. Our results indicate that the migrating neuroblasts were not replenished, despite the presence of type B cells in the SVZ post-irradiation. This study provides novel insights into the effects of localized SVZ radiation on neurogenesis and cell migration that may potentially lead to the development of new radiotherapy strategies to minimize damage to NSCs and neuroblast migration.

A radiotherapy technique to limit dose to neural progenitor cell niches without compromising tumor coverage.

Radiation therapy (RT) for brain tumors is associated with neurocognitive toxicity which may be a result of damage to neural progenitor cells (NPCs). We present a novel technique to limit the radiation dose to NPC without compromising tumor coverage. A study was performed in mice to examine the rationale and another was conducted in humans to determine its feasibility. C57BL/6 mice received localized radiation using a dedicated animal irradiation system with on-board CT imaging with either: (1) Radiation which spared NPC containing regions; (2) Radiation which did not spare these niches; or (3) Sham irradiation. Mice were sacrificed 24 h later and the brains were processed for immunohistochemical Ki-67 staining. For the human component of the study, 33 patients with primary brain tumors were evaluated. Two intensity modulated radiotherapy (IMRT) plans were retrospectively compared: a standard clinical plan and a plan which spares NPC regions while maintaining the same dose coverage of the tumor. The change in radiation dose to the contralateral NPC-containing regions was recorded. In the mouse model, non-NPC-sparing radiation treatment resulted in a significant decrease in the number of Ki67(+) cells in dentate gyrus (DG) (P = 0.008) and subventricular zone (SVZ) (P = 0.005) compared to NPC-sparing radiation treatment. In NPC-sparing clinical plans, NPC regions received significantly lower radiation dose with no clinically relevant changes in tumor coverage. This novel radiation technique should significantly reduce radiation doses to NPC containing regions of the brain which may reduce neurocognitive deficits following RT for brain tumors.

Biological horizons for targeting brain malignancy.

Though currently available clinical treatments and therapies have clearly extended the survival of patients with brain tumors, many of these advances are short lived, particularly with respect to high grade gliomas such as glioblastoma multiforme. The missing link to an efficacious treatment of high grade gliomas is a more complete understanding of the basic molecular and cellular origin of brain tumors. However, new discoveries of stem cell and developmental neurobiology have now borne the cancer stem cell hypothesis, drawing off of intriguing similarities between benign and malignant cells within the central nervous system. Investigation of cancer stem cell hypothesis and brain tumor propagation is the current frontier of stem cell and cancer biology. Neurosurgery is also watching closely this promising new area of focus. "Molecular neurosurgery", glioma treatments involving biologics using neural stem cells to target the cancer at the level of individual migratory cell, is a rapidly evolving field. This coming progression of applied cancer stem cell research, coupled with current modalities, promises more comprehensive brain cancer interventions.

Discovery of a Covalent Inhibitor of KRASG12C (AMG 510) for the Treatment of Solid Tumors.

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).

Discovery of N-(1-Acryloylazetidin-3-yl)-2-(1H-indol-1-yl)acetamides as Covalent Inhibitors of KRASG12C.

KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C, which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRASG12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRASG12C with submicromolar inhibition of downstream signaling in a KRASG12C-specific manner.

Gene expression changes in the rodent hippocampus following whole brain irradiation.

Therapeutic cranial irradiation may result in debilitating cognitive impairments. In human patients these deficits are age and radiation dose-dependent and are attributed to a diminished capability to learn and memorize new tasks and information. Because of the known involvement of the hippocampus in memory consolidation, it is important to identify irradiation-induced changes including alterations in gene expression in this structure. Whole brain irradiation doses of 0, 0.3, 3, 10, or 30 Gray (Gy) were administered to 3-month-old rats in a single session. Twenty-four hours following cranial irradiation, hippocampi were processed for oligonucleotide microarrays analysis. Metallothioneins (MT)-I and -II, heat shock protein (Hsp-27), glial fibrillary acidic protein alpha (GFAP), and c-Fos genes were altered significantly across the various doses of irradiation. A pathway analysis shows that these genes were centered around the immediate early gene myc and tumor suppressor gene (TP53). Our results identified important genes and possible pathways that are altered in the hippocampus in the acute phase following cranial irradiation, and implicate gene pathways important for both learning and memory and apoptosis.

Somatic retrotransposition is infrequent in glioblastomas.

BACKGROUND: Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals. RESULTS: We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions. CONCLUSIONS: These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.

Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes.

Glial progenitor cells (GPCs) are a potential source of malignant gliomas. We used A2B5-based sorting to extract tumorigenic GPCs from human gliomas spanning World Health Organization grades II-IV. Messenger RNA profiling identified a cohort of genes that distinguished A2B5+ glioma tumor progenitor cells (TPCs) from A2B5+ GPCs isolated from normal white matter. A core set of genes and pathways was substantially dysregulated in A2B5+ TPCs, which included the transcription factor SIX1 and its principal cofactors, EYA1 and DACH2. Small hairpin RNAi silencing of SIX1 inhibited the expansion of glioma TPCs in vitro and in vivo, suggesting a critical and unrecognized role of the SIX1-EYA1-DACH2 system in glioma genesis or progression. By comparing the expression patterns of glioma TPCs with those of normal GPCs, we have identified a discrete set of pathways by which glial tumorigenesis may be better understood and more specifically targeted.

Gliomagenesis and the use of neural stem cells in brain tumor treatment.

The role of neural stem cells (NSCs) in both the physiological and pathological processes in the brain has been refined through recent studies within the neuro-oncological field. Alterations in NSC regulatory mechanisms may be fundamental for the development and progression of malignant gliomas. A subpopulation of cells within the tumor known as brain tumor stem cells (BTSCs) have been shown to share key properties with NSCs. The BTSC hypothesis has significantly contributed to a potential understanding as to why brain tumors hold such dismal prognosis. On the other hand, the normal NSCs possess the capacity to migrate extensively towards the tumor bulk as well as to lingering neoplastic regions of the brain. The tropism of NSCs towards brain tumors may provide an additional tool for the treatment of brain cancer. The creation of potential therapies through the use of NSCs has been studied and includes the delivery of gene products to specific locations of the central nervous system selectively targeting malignant brain tumor cells and maximizing the efficiency of their delivery. Here, the proposed mechanisms of how brain tumors emerge, the molecular pathways interrupted in NSC pathogenesis and the most recent preclinical results in the use of NSCs for glioma treatment are reviewed.

Ionizing radiation impairs the formation of trace fear memories and reduces hippocampal neurogenesis.

Long-term cognitive impairments are a feared consequence of therapeutic cranial irradiation in children as well as adults. Studies in animal models suggest that these deficits may be associated with a decrease in hippocampal granule cell proliferation and survival. In the present study the authors examined whether whole brain irradiation would affect trace fear conditioning, a hippocampal-dependent task. Preadolescent (postnatal Day 21, PD 21), adolescent (PD 50), and postadolescent (PD 70) rats received single doses of 0 Gray (Gy), 0.3 Gy, 3 Gy, or 10 Gy whole brain irradiation. Three months after radiation treatment, a significant dose-dependent decrease in bromo-deoxyuridine positive cells was observed. Irradiation produced a dose-dependent decrease in freezing in response to the conditioned stimulus in all age groups. Interestingly, the authors found no differences in context freezing between irradiated and control groups. Further, there were no differences in delay fear memories, which are independent of hippocampus function. Our results strongly indicate that irradiation impairs associative memories dependent on hippocampus and this deficit is accompanied by a decrease in granule cell neurogenesis indicating that these cells may be involved in normal hippocampal memory function.

A histology-based atlas of the C57BL/6J mouse brain deformably registered to in vivo MRI for localized radiation and surgical targeting.

The C57BL/6J laboratory mouse is commonly used in neurobiological research. Digital atlases of the C57BL/6J brain have been used for visualization, genetic phenotyping and morphometry, but currently lack the ability to accurately calculate deviations between individual mice. We developed a fully three-dimensional digital atlas of the C57BL/6J brain based on the histology atlas of Paxinos and Franklin (2001 The Mouse Brain in Stereotaxic Coordinates 2nd edn (San Diego, CA: Academic)). The atlas uses triangular meshes to represent the various structures. The atlas structures can be overlaid and deformed to individual mouse MR images. For this study, we selected 18 structures from the histological atlas. Average atlases can be created for any group of mice of interest by calculating the mean three-dimensional positions of corresponding individual mesh vertices. As a validation of the atlas' accuracy, we performed deformable registration of the lateral ventricles to 13 MR brain scans of mice in three age groups: 5, 8 and 9 weeks old. Lateral ventricle structures from individual mice were compared to the corresponding average structures and the original histology structures. We found that the average structures created using our method more accurately represent individual anatomy than histology-based atlases alone, with mean vertex deviations of 0.044 mm versus 0.082 mm for the left lateral ventricle and 0.045 mm versus 0.068 mm for the right lateral ventricle. Our atlas representation gives direct spatial deviations for structures of interest. Our results indicate that MR-deformable histology-based atlases represent an accurate method to obtain accurate morphometric measurements of a population of mice, and that this method may be applied to phenotyping experiments in the future as well as precision targeting of surgical procedures or radiation treatment.

Intra-operatively obtained human tissue: protocols and techniques for the study of neural stem cells.

The discoveries of neural (NSCs) and brain tumor stem cells (BTSCs) in the adult human brain and in brain tumors, respectively, have led to a new era in neuroscience research. These cells represent novel approaches to studying normal phenomena such as memory and learning, as well as pathological conditions such as Parkinson's disease, stroke, and brain tumors. This new paradigm stresses the importance of understanding how these cells behave in vitro and in vivo. It also stresses the need to use human-derived tissue to study human disease because animal models may not necessarily accurately replicate the processes that occur in humans. An important, but often underused, source of human tissue and, consequently, both NSCs and BTSCs, is the operating room. This study describes in detail both current and newly developed laboratory techniques, which in our experience are used to process and study human NSCs and BTSCs from tissue obtained directly from the operating room.