Travis Fleming
@tjflemin.bsky.social
Harvard Medical School PhD '25 @bloodgenes.bsky.social lab
Functional genomics, CRISPR screens, Acute Myeloid Leukemia, Drug discovery, targeted protein degradation
@danafarber.bsky.social
@bostonchildrens.bsky.social
@broadinstitute.org
Functional genomics, CRISPR screens, Acute Myeloid Leukemia, Drug discovery, targeted protein degradation
@danafarber.bsky.social
@bostonchildrens.bsky.social
@broadinstitute.org
Strikingly, activating this cisRE significantly impaired the engraftment ability of primary AML cells, highlighting the therapeutic potential of reactivating myeloid differentiation programs to disrupt the fitness of stem cell-like leukemia cells.
January 2, 2025 at 2:47 PM
Strikingly, activating this cisRE significantly impaired the engraftment ability of primary AML cells, highlighting the therapeutic potential of reactivating myeloid differentiation programs to disrupt the fitness of stem cell-like leukemia cells.
Finally, we transplanted these cells into immunodeficient mice to assess how CEBPA cisRE activation impacted leukemia burden and engraftment of modified cells.
January 2, 2025 at 2:47 PM
Finally, we transplanted these cells into immunodeficient mice to assess how CEBPA cisRE activation impacted leukemia burden and engraftment of modified cells.
The approach yielded a modest increase in CEBPA expression itself. However, this transient and subtle activation was sufficient to induce significant differentiation phenotypes during ex vivo culture.
January 2, 2025 at 2:47 PM
The approach yielded a modest increase in CEBPA expression itself. However, this transient and subtle activation was sufficient to induce significant differentiation phenotypes during ex vivo culture.
We next assessed if activation of this cisRE alone was sufficient to induce differentiation of these primary AMLs. Here, we delivered CRISPRa mRNA and the same guide RNAs targeting this cisRE.
January 2, 2025 at 2:47 PM
We next assessed if activation of this cisRE alone was sufficient to induce differentiation of these primary AMLs. Here, we delivered CRISPRa mRNA and the same guide RNAs targeting this cisRE.
Remarkably, across a panel of patient samples, MECOM KO induced significant loss of stem cell-like leukemia cells, while inactivation of the CEBPA cisRE could almost completely rescue this phenotype and maintain cells in more stem cell-like states.
January 2, 2025 at 2:47 PM
Remarkably, across a panel of patient samples, MECOM KO induced significant loss of stem cell-like leukemia cells, while inactivation of the CEBPA cisRE could almost completely rescue this phenotype and maintain cells in more stem cell-like states.
Nonetheless, we wanted to determine if the functional link between MECOM and this CEBPA cisRE was conserved in primary AML samples. To do so, we knocked out MECOM and co-inactivated this cisRE, hypothesizing that cisRE inactivation should rescue MECOM-KO-induced differentiation.
January 2, 2025 at 2:47 PM
Nonetheless, we wanted to determine if the functional link between MECOM and this CEBPA cisRE was conserved in primary AML samples. To do so, we knocked out MECOM and co-inactivated this cisRE, hypothesizing that cisRE inactivation should rescue MECOM-KO-induced differentiation.
We then performed an orthogonal screen using CRISPRa, asking if in the absence of MECOM perturbation, could activation of any single cisRE be sufficient to induce myeloid differentiation? Surprisingly, the only hit from this screen was the same cisRE linked to CEBPA!
January 2, 2025 at 2:47 PM
We then performed an orthogonal screen using CRISPRa, asking if in the absence of MECOM perturbation, could activation of any single cisRE be sufficient to induce myeloid differentiation? Surprisingly, the only hit from this screen was the same cisRE linked to CEBPA!
We first leveraged a CRISPRi screen to determine if repressing any single cisRE is sufficient to maintain cells in a CD34+ stem cell-like state, even after MECOM degradation. The most significant hit from this screen was a cisRE 42 kb away from the myeloid TF CEBPA.
January 2, 2025 at 2:47 PM
We first leveraged a CRISPRi screen to determine if repressing any single cisRE is sufficient to maintain cells in a CD34+ stem cell-like state, even after MECOM degradation. The most significant hit from this screen was a cisRE 42 kb away from the myeloid TF CEBPA.
But which parts of these MECOM-regulated networks are functionally important? This motivated us to perform functional genomic screens to identify MECOM controlled cisREs that are essential in facilitating MECOM’s ability to block differentiation in stem cell-like leukemia cells.
January 2, 2025 at 2:47 PM
But which parts of these MECOM-regulated networks are functionally important? This motivated us to perform functional genomic screens to identify MECOM controlled cisREs that are essential in facilitating MECOM’s ability to block differentiation in stem cell-like leukemia cells.
We hypothesized that if our gene and cisRE networks are repressed in primary AML, MECOM-driven stem cell-like states would be anti-correlated with their activity. Indeed, analyses of single cell genomics data of a large AML patient cohort confirmed this hypothesis.
January 2, 2025 at 2:47 PM
We hypothesized that if our gene and cisRE networks are repressed in primary AML, MECOM-driven stem cell-like states would be anti-correlated with their activity. Indeed, analyses of single cell genomics data of a large AML patient cohort confirmed this hypothesis.
We defined conserved networks of genes and cis-regulatory elements (cisREs) under the direct repression of MECOM. However, we ultimately wanted to determine whether these MECOM-repressed gene and cisRE networks were relevant in primary AMLs.
January 2, 2025 at 2:47 PM
We defined conserved networks of genes and cis-regulatory elements (cisREs) under the direct repression of MECOM. However, we ultimately wanted to determine whether these MECOM-repressed gene and cisRE networks were relevant in primary AMLs.
We then profiled the direct molecular changes that occur upon MECOM loss. Interestingly, we observed significant increases in gene expression and chromatin accessibility hours after degradation, suggesting MECOM functions predominantly as a transcriptional repressor in AML.
January 2, 2025 at 2:47 PM
We then profiled the direct molecular changes that occur upon MECOM loss. Interestingly, we observed significant increases in gene expression and chromatin accessibility hours after degradation, suggesting MECOM functions predominantly as a transcriptional repressor in AML.
Notably, MECOM degradation resulted in striking myeloid differentiation phenotypes and eventually cell death, presumably due to the loss of stem-like, self-renewal capabilities.
January 2, 2025 at 2:47 PM
Notably, MECOM degradation resulted in striking myeloid differentiation phenotypes and eventually cell death, presumably due to the loss of stem-like, self-renewal capabilities.
To characterize MECOM’s direct function, in the absence of confounding cell-state alterations, we engineered a suite of AML cell lines with an endogenous MECOM-FKBP12F36V degron. These models enable rapid and specific degradation of all MECOM protein in AML cells within minutes!
January 2, 2025 at 2:47 PM
To characterize MECOM’s direct function, in the absence of confounding cell-state alterations, we engineered a suite of AML cell lines with an endogenous MECOM-FKBP12F36V degron. These models enable rapid and specific degradation of all MECOM protein in AML cells within minutes!
As shown by Ruud Delwel and co, HSC gene expression programs in AML are frequently driven by increased expression of MECOM, a TF that plays a key role in normal HSC maintenance. However, the mechanisms by which MECOM drives stem cell-like, high-risk features remained unclear.
January 2, 2025 at 2:47 PM
As shown by Ruud Delwel and co, HSC gene expression programs in AML are frequently driven by increased expression of MECOM, a TF that plays a key role in normal HSC maintenance. However, the mechanisms by which MECOM drives stem cell-like, high-risk features remained unclear.
For some AMLs, therapies like retinoids and menin inhibitors can induce differentiation and improve patient outcomes. But there is still a significant lack of targeted approaches to induce therapeutic differentiation of some of the most primitive AMLs with the worst prognoses.
January 2, 2025 at 2:47 PM
For some AMLs, therapies like retinoids and menin inhibitors can induce differentiation and improve patient outcomes. But there is still a significant lack of targeted approaches to induce therapeutic differentiation of some of the most primitive AMLs with the worst prognoses.
As demonstrated by John Dick/Andy Zeng and other colleagues, HSC gene expression programs in AML drive primitive cell states linked to poor patient prognosis and high risk of relapse.
January 2, 2025 at 2:47 PM
As demonstrated by John Dick/Andy Zeng and other colleagues, HSC gene expression programs in AML drive primitive cell states linked to poor patient prognosis and high risk of relapse.
Stem cell-like states confer poor outcomes in blood cancer—but what mechanisms drive this and how can they be therapeutically targeted? In our new preprint, we show how a single TF represses one enhancer to maintain a subset of high-risk leukemias: 🧵👇
www.biorxiv.org/content/10.1...
www.biorxiv.org/content/10.1...
January 2, 2025 at 2:47 PM
Stem cell-like states confer poor outcomes in blood cancer—but what mechanisms drive this and how can they be therapeutically targeted? In our new preprint, we show how a single TF represses one enhancer to maintain a subset of high-risk leukemias: 🧵👇
www.biorxiv.org/content/10.1...
www.biorxiv.org/content/10.1...
Roll out of the hotel bed at 5pm on Monday and come listen my talk! Our CRISPR screening to elucidate mechanisms of #AML stem cell maintenance is guaranteed to calm your throbbing headache. @ash-hematology.bsky.social
December 5, 2024 at 12:30 AM
Roll out of the hotel bed at 5pm on Monday and come listen my talk! Our CRISPR screening to elucidate mechanisms of #AML stem cell maintenance is guaranteed to calm your throbbing headache. @ash-hematology.bsky.social