Peer review of: MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
This manuscript describes a potential resistance mechanism in temozolomide-treated glioblastoma
(GBM). In a genomic profiling of recurrent GBMs, the authors identify fusions involving the DNA
repair enzyme MGMT, associated with a small percentage of tumors. They find that MGMT fusions
are mutually exclusive with MGMT hypomethylation and global hypermutation, other established
resistance mechanisms. They go on to perform functional analysis in U251 an U87 cells. They find
that expression of MGMT fusions increases MGMT expression and decreases sensitivity to
temozolomide, and that these phenomena are correlated. Moreover, MGMT fusion expression
increases temozolomide resistance in a murine xenograft model. Finally, they demonstate that
MGMT fusions are shed in exosomes, and can be detected in peripheral blood, pointing to a
potential method for clinical screening.
While the small number of MGMT-fused cases does detract somewhat from the significance of this
 work, it is a well-performed study describing a novel mechanisms of resistance in a deadly tumor
 type.
 My comments are below.
1) The reliance of the author’s functional analyses on U87 and U251 cells is somewhat
problematic. These lines have been subjected to extensive culture over the years and may not
represent the best models of glioma biology. Many patient-derived glioma stem cells (GSCs) are
currently available and recapitulate the broad spectrum of molecular features characterizing
gliomas in general. The authors should recapitulate their key in vitro and in vivo findings using
GSCs. The authors might also consider using the RCAS/tv-a system, which they have leveraged
very effectively in the past, the address some of their hypotheses in vivo.
2) Recurrent glioma specimens are notoriously heterogeneous in their composition with regard to
tumor content, reactive tissue, necrotizing treatment effect, and inflammatory cells. This
heterogeneity could impact the detection of genomic factors associated with resistance, including
MGMT-fusions. Can the authors provide more information as to the histopathological and molecular
QC measures used in their profiling studies? Was there a tumor content threshold? Who did the
analysis? What were the purity/ploidy measurements of their samples?
More minor
1) In the introduction, the authors should better explain the distinction between IDH-wild type and
IDH-mutant glioma. General readers will not appreciate this difference. They should indicate early
in the paper that malignant gliomas come in two basic varieties, IDH-mutant and IDH-wild type,
and both are treated with temozolomide as the standard of care.
2) On a related note, the authors should consider presenting their genomic data more in the
 context of IDH-mutant vs IDH-wild type glioma, rather than HGG vs LGG.
 Reviewer #2 (Remarks to the Author):
The manuscript “MGMT Genomic rearrangements...” submitted by Oldrini et al describes studies
addressing a new form of MGMT genetic alteration in glioma that may contribute to TMZ
resistance. In this study the authors show that in a small percentage of these patients, perhaps
3%, fusions of the MGMT gene with the 5’ UTR of other genes results in higher level MGMT
expression that in turn appears associated with TMZ resistance. In terms of novelty this is an
interesting and unique finding, and although as the authors note a similar MGMT fusion has been
 noted in a single medulloblastoma, the full range of such fusions in glioma has not been previously
 described. The work is also of interest in that there is growing interest in promoter highjacking and
 fusion transcripts as drivers in cancer, although this mechanism has not in the past been shown to
 involve MGMT. The work is also of importance as TMZ remains a mainline therapeutic for most
 forms of glioma, and resistance to TMZ and recurrence is common. As such the identification of a
 new mechanism of resistance and possibly a biomarker of this resistance, if even in a small
 percentage of patients is of interest to a broad range of investigators.
 In terms of the studies themselves, they appear carefully done, appropriately controlled, and
 adequately statistically evaluated. There are no instances in which the data presented do not
 support the conclusions reached. I have only a few questions regarding the work as presented:
 1. The authors note that they have analyzed “hypermutation” in the samples and found that it was
 mutually exclusive with MGMT fusion. It is not clear however what type of “hypermutation” the
 authors are considering. Is this hypermutation the global type of hypermutation noted in TMZ-
 treated recurrent GBM or are the authors talking about MGMT hypermutation? If the latter, is this
 MGMT gene or promoter hypermutation? One really cant tell from the text or from the description
 provided in the methods section. This is an important point as most readers would be interested in
 whether MGMT fusion was mutually exclusive with TMZ-induced genomic hypermutation (as one
 might expect it to be).
 2. Im also a little unclear about the generation of the cells carrying MGMT fusions by CRISPR. For
 such experiments one would typically use CRISPR-Cas to create clones, then sort through the
 clones to find those with the desired recombination in one or both copies of the targeted genes.
 Line 122 in the text does not clearly describe how the process was carried out, and the suggestion
 that the end result of the initial engineering was “a mixed population of cells carrying different
 fusion events” is puzzling. Additionally the further use of populations of cells that arose as a result
 of TMZ selection is also a little puzzling as one would expect all the appropriately engineered cells
 to be TMZ resistant. This entire creation/selection process needs to be better described.