Summary: MAPK pathway activation and oncogene-induced senescence

pLGG is described as predominantly a single-pathway disease in which somatic or germline alterations converge on constitutive activation of the RAS/RAF/MEK/ERK (MAPK) pathway. KIAA1549::BRAF fusions arise from a tandem duplication at 7q34 that removes the N-terminal regulatory domain of BRAF, producing RAS-independent, dimeric RAF signaling. BRAF p.V600E acts as a phosphomimetic that produces constitutive monomeric kinase activity. Germline NF1 loss removes a negative regulator of RAS. Tumors without an identified driver show similar levels of MAPK pathway up-regulation. Sustained MAPK activation also induces oncogene-induced senescence mediated by p16(INK4a) and the senescence-associated secretory phenotype, which was found in 46 of 52 pilocytic astrocytomas (88.5%) and is proposed to explain slow growth and rare progression. CDKN2A deletion, which co-occurs with BRAF p.V600E, is associated with escape from senescence. Type I BRAF inhibitors are limited to BRAF V600E tumors because they can paradoxically activate MAPK signaling in RAF fusion-driven tumors.

BRAF fusion: loss of the BRAF regulatory domain and RAS-independent dimeric signaling

“This rearrangement resulted in the N-terminal regulatory domain of BRAF being lost, leading to downstream up-regulation of the RAS/MAPK signaling pathway” (opens the source at this quote in a new tab)

“BRAF fusion-positive tumors demonstrate constitutive, RAS-independent, dimeric RAF signaling, resulting in sustained MAPK pathway activation.” (opens the source at this quote in a new tab)
“In contrast, activating BRAF point mutations, most commonly BRAF p.V600E (15–20% of pLGG), result in constitutive monomeric kinase activation and define a biologically and clinically distinct subgroup” (opens the source at this quote in a new tab)

“This gene alteration results in the expression of a wild-type BRAF catalytic domain without its normal regulatory domain, thereby rendering constitutively active BRAF activity.” (opens the source at this quote in a new tab)

BRAF V600E: constitutive kinase activation

“Mutations in BRAF, primarily in which a valine is replaced with a glutamic acid at position 600 (p.V600E), act as a phosphomimetic within the RAS/MAPK pathway, rendering it constitutively active” (opens the source at this quote in a new tab)

Single-pathway disease: MAPK up-regulation including tumors without an identified driver

“Pediatric low-grade glioma is predominantly a single pathway disease driven by alterations in the mitogen-activated protein kinase (MAPK) signaling pathway (also known as the RAS-RAF-MEK-ERK pathway).” (opens the source at this quote in a new tab)

“Interestingly, all tumors had significantly increased ppERK as compared to normal brain controls (p<0.0001, ANOVA) (Figure 3A).” (opens the source at this quote in a new tab)
“indicating similar levels of RAS/MAPK up-regulation in pLGG lacking a clear molecular driver” (opens the source at this quote in a new tab)

NF1: loss of neurofibromin as a negative regulator of RAS

“NF1 is caused by a germline mutation in the NF1 tumor suppressor gene, which encodes neurofibromin, a GTPase-activating protein that functions as a negative regulator RAS” (opens the source at this quote in a new tab)

Oncogene-induced senescence in pilocytic astrocytoma

“Our results indicate that PAs are senescent as evidenced by marked senescence-associated acidic β-galactosidase activity, low KI-67 index, and induction of p16(INK4a) but not p53 in the majority of 52 PA samples (46 of 52; 88.5%).” (opens the source at this quote in a new tab)
“OIS may account for the slow growth pattern in PA, the lack of progression to higher-grade astrocytomas, and the high overall survival of affected patients.” (opens the source at this quote in a new tab)

“BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation.” (opens the source at this quote in a new tab)
“Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16 INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16 INK4a had significantly shorter overall survival.” (opens the source at this quote in a new tab)

Senescence-associated secretory phenotype

“MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas.” (opens the source at this quote in a new tab)
“We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator.” (opens the source at this quote in a new tab)
“High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival.” (opens the source at this quote in a new tab)

“While pLGG harbors fewer somatic driver alterations relative to other cancers, they also harbor significant transcriptional heterogeneity, with cells expressing MAPK programs, cell-cycle programs, and senescence programs.” (opens the source at this quote in a new tab)

CDKN2A loss and escape from senescence

“In PLGGs, CDKN2A loss has been reported to be associated with escape from oncogene-induced senescence, especially when combined with BRAF mutations.” (opens the source at this quote in a new tab)

“This is in line with reports showing that co-occurrence of CDKN2A deletion with BRAF p.V600E is associated with escape from oncogene-induced senescence” (opens the source at this quote in a new tab)

Paradoxical MAPK activation by type I BRAF inhibitors in RAF fusion-driven tumors

“The use of type I BRAF inhibitors is limited to patients with tumors harboring a BRAF V600E mutation due to the risk of paradoxical activation of the MAPK pathway and accelerated tumor growth if used in patients with tumors harboring a RAF fusion, such as those involving BRAF or CRAF/RAF1” (opens the source at this quote in a new tab)

Effect of prolonged MAPK inhibition on senescence

“The impact of prolonged MAPK pathway inhibition on oncogene-induced senescence (OIS) in pLGG remains unknown and it represents an important gap in understanding what could be the long-term biological consequences of targeted therapies.” (opens the source at this quote in a new tab)

Absence of late progression-related death as clinical evidence for oncogene-induced senescence

“Indeed, none of the NF1 patients died in adulthood, and none of the other PLGG adult survivors in this study died of tumor progression.” (opens the source at this quote in a new tab)
“These intriguing observations support the current biological hypothesis that in RAS/MAPK-driven tumors, oncogene-induced senescence causes spontaneous growth arrest and tumor burnout in adolescent and young adults with PLGG.” (opens the source at this quote in a new tab)

Natural history of pilocytic astrocytoma as cycles of growth and dormancy

“The natural history of PA is unique, as these tumors are self-containing, almost never progress to higher-grade astrocytomas, and their initial growth is usually followed by a decrease in tumor activity, after which they enter a dormant phase and either remain quiescent or restart cycle (s) of growth followed by dormancy, similar to what is observed in OIS in vitro.” (opens the source at this quote in a new tab)
“Moreover, artificial acceleration of this OIS program could present an interesting therapeutic possibility in patients with progressive disease.” (opens the source at this quote in a new tab)

Senescence-associated secretory phenotype expression and progression-free survival by resection status

“We here show that a high SASP sum score is associated with favorable progression-free survival (PFS) independent of tumor resection status. We identify two distinct patient cohorts, one with high or intermediate SASP and complete resection (100% 3-year PFS), and one with low SASP and incomplete resection (0% 3-year PFS).” (opens the source at this quote in a new tab)

Non-response of senescent tumor cells to chemotherapy and MEK inhibition

“No significant response to carboplatin, vincristine, or trametinib was detected in senescent DKFZ-BT66 cells” (opens the source at this quote in a new tab)
“In conclusion, addition of a BCL2 family inhibitor to the standard-of-care treatment regimen for patients with pilocytic astrocytoma could help eradicate senescent pilocytic astrocytoma cells, which do not respond to chemotherapy and might be the source of tumor recurrence or progression at a later time point.” (opens the source at this quote in a new tab)

Reversal of growth arrest by anti-inflammatory treatment

“Treatment with the broad anti-inflammatory drug dexamethasone, however, induced proliferation, exemplary inhibition of the IL1 pathway, and suppression of the SASP in DKFZ-BT66 cells in OIS.” (opens the source at this quote in a new tab)
“As dexamethasone is a drug commonly used in pediatric clinical practice, this result is potentially of high clinical relevance.” (opens the source at this quote in a new tab)
“The SASP can induce growth arrest in proliferating pilocytic astrocytoma cells, while suppression of the SASP by anti-inflammatory treatment leads to bypass of growth arrest.” (opens the source at this quote in a new tab)

Determinants of the induction and level of oncogene-induced senescence

“Limiting signals affecting induction and levels of OIS may be dictated not only by the initiating genetic alteration but also by the threshold (intensity and duration) of the signaling and/or the tissue type (cell of origin, microenvironment including the stroma and immune response).” (opens the source at this quote in a new tab)
“This can explain the dichotomy between activation of an oncogenic pathway, which was recently found to be constitutively activated in 75% to 100% of PAs, and their tendency toward growth arrest.” (opens the source at this quote in a new tab)

MEK inhibition in the senescence model and the limits of a single cell line

“An increase in metabolic activity in both proliferating and senescent DKFZ-BT66 cells was observed under trametinib treatment, as we have described previously” (opens the source at this quote in a new tab)
“The current lack of appropriate models beyond DKFZ-BT66 prevents reproduction in a second cell line, and as such represents a limitation of this study, highlighting the urgent need for new additional models at the same time.” (opens the source at this quote in a new tab)
“Follow-up studies are warranted to assess the clinical impact of the use of anti-inflammatory (and immunosuppressive) drugs such as, for example, dexamethasone in pediatric patients with pilocytic astrocytoma.” (opens the source at this quote in a new tab)

Tovorafenib (DAY101)

Pathophysiology