Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen
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Abstract
Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9 -driven leukemia.
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###This manuscript is in revision at eLife
The manuscript was reviewed by Review Commons. eLife's decision letter, sent to the authors on April 18, 2020, follows.
Summary
In this manuscript, the authors study the transcriptional regulation of HOXA9, a transcription factor that plays a central role in homeostasis of immature hematopoietic cell types and in the development of leukemia. They use the CRISPR/Cas9 technique to introduce a fluorescence reporter cassette into the endogenous HOXA9 locus of a human MLL/AF4-rearranged B-ALL cell line. After validating this engineered cell line, they perform multiple genetic screens to identify potential transcriptional regulators of HOXA9 and to delineate essential transcription factors in this cell line. They identify USF2 as new transcription factor that modulates expression of HOXA9.
Major …
###This manuscript is in revision at eLife
The manuscript was reviewed by Review Commons. eLife's decision letter, sent to the authors on April 18, 2020, follows.
Summary
In this manuscript, the authors study the transcriptional regulation of HOXA9, a transcription factor that plays a central role in homeostasis of immature hematopoietic cell types and in the development of leukemia. They use the CRISPR/Cas9 technique to introduce a fluorescence reporter cassette into the endogenous HOXA9 locus of a human MLL/AF4-rearranged B-ALL cell line. After validating this engineered cell line, they perform multiple genetic screens to identify potential transcriptional regulators of HOXA9 and to delineate essential transcription factors in this cell line. They identify USF2 as new transcription factor that modulates expression of HOXA9.
Major Revisions
If the authors can commit to adding the following data, as they indicate in their rebuttal, the manuscript would be greatly strengthened and could be considered acceptable:
The authors should include their data on the independent loss-of-function CRISPR transcription factor screen in SEM HOXA9-P2A-mCherry MLLr reporter line ectopically expressing HOXA9-MEIS1 to overcome the possibility that key regulators could be missed in the CRISPR/Cas9 screen due to survival dropout.
The authors should include supporting data for the key observations in the manuscript in other cell lines; for example, as indicated by the authors, the data gathered using an additional HOXA9 MLLr AML reporter cell line established in OCI-AML2 cells to further support findings from the initial in SEM MLLr ALL reporter line.
The authors should perform USF2 knockout experiments in multiple non-MLLr cell lines according to the reviewer's suggestions. As an example, the authors should repeat the competitive proliferation assay to determine the effects of the single knockout of USF1 and USF2 vs the double KO in SEM cells and other MLLr leukemia cell lines with proper controls.
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