Supplementary Table 1 lists oligonucleotide sequences and cell line information

Supplementary table 4 lists ETV6-regulated gene-enhancer linkages

Contains Supplementary Figures 1-12 and additional information about Supplementary tables

Supplementary Table 3 summarizes TF ChIP-Seq results and motif enrichment statistics

Supplementary Table 2 details known and de novo motif analysis for distal acetylation clusters

Supplementary Table 5 annotates genome-wide candidate ETV6-repressed enhancers

Supplementary Table 6 details GGAA repeat overlap analysis of new and previously published ETS factor datasets.

There is an urgent need to find targeted agents for T cell acute lymphoblastic leukemia (T-ALL). NOTCH1 is the most frequently mutated oncogene in T-ALL, but clinical trials showed that pan-Notch inhibitors caused dose-limiting toxicities. Thus, we shifted our focus to ETS1, which is one of the transcription factors that most frequently co-bind Notch-occupied regulatory elements in the T-ALL context. To identify the most essential enhancers, we performed a genome-wide CRISPRi screen of the strongest ETS1-dependent regulatory elements. The top-ranked element is located in an intron of AHI1 that interacts with the MYB promoter and is amplified with MYB in approximately 8.5% of patients with T-ALL. Using mouse models, we showed that this enhancer promoted self-renewal of hematopoietic stem cells and T cell leukemogenesis, maintained early T cell precursors, and restrained myeloid expansion with aging. We named this enhancer the hematopoietic stem cell MYB enhancer (H-Me). The H-Me showed limited activity and function in committed T cell progenitors but was accessed during leukemogenesis. In one T-ALL context, ETS1 bound the ETS motif in the H-Me to recruit cBAF to promote chromatin accessibility and activation. ETS1 or cBAF degraders impaired H-Me function. Thus, we identified a targetable stem cell element that was co-opted for T cell transformation.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid malignancy derived from immature T cells. No targeted therapies are currently available. Although over 60% of T-ALL samples carry activating mutations in the Notch signaling pathway, this finding has been difficult to translate clinically as pan-Notch inhibitors have significant toxicities. We previously showed that ETS1 is an important Notch co-binding transcription factor in T-ALL and could represent a safer therapeutic target. ETS motifs are the top-ranked motif next to Notch-RBPJ sites in T-ALL, suggesting a strong collaborative interaction. ETS1 knockdown or deletion impairs T-ALL growth in T-ALL cell lines, patient-derived xenografts, and genetically engineered mouse models, illustrating its importance in T-ALL cell growth. In contrast to Notch inhibition, ubiquitous deletion of Ets1 in murine models does not appear to cause significant toxicity. To further define the downstream enhancers and oncogenes comprising the “regulome” of ETS1 in T-ALL, we used chromatin profiling data to perform a CRISPRi essentiality screen directed at high-probability ETS1-dependent regulatory elements in the human T-ALL cell line THP-6. The “Activity” model (Fulco et al.) was used to predict highly active regulatory regions. Further criteria included “dynamic” ETS1 binding sites (decreased H3K27Ac and ETS1 signals upon ETS1 knockdown at FDR<0.05) and high H3K27Ac ChIP-seq signal in primary T-ALL samples. This library was transduced into THP-6 cells that express doxycycline inducible dCas9-KRAB. Genomic DNA was sequenced before and after expansion in doxycycline. MAGeCK algorithm was used to score elements for negative selection relative to non-targeting controls. Our screen identified 9 regulatory elements with FDR < 6.0E05 that were highly negatively selected and within -LFC range of pan-essential genes. Notably, we identified a previously uncharacterized ETS1-dependent MYB enhancer as the #1 most negatively selected ETS1-dependent T-ALL regulatory element. Characterization and functional analysis of this enhancer is being reported in a separate abstract at this meeting. 2 other highly negatively selected elements were well-characterized T-ALL enhancers. These included the Notch-MYC enhancer (“N-Me”), which we previously described as ETS1-dependent, and a T-ALL enhancer linked to BCL11B. These data validate the utility of our screen. We additionally identified three novel regulatory elements that have not been previously linked to T-ALL pathogenesis. One notable element is an intronic enhancer linked by H3K27Ac Hi-ChIP to ETS1, FLI1, and an uncharacterized lncRNA. Repression of this +17kB ETS1 intronic enhancer with targeted CRISPRi suppressed expression of ETS1 by 2-fold and inhibited growth by 3-to-4-fold in THP-6 cells. Thus, this enhancer may participate in positive auto-regulation. We next identified a long-range enhancer linked to the promoter of MSI2 by H3K27Ac Hi-ChIP. MSI2 is a recently described T-ALL oncogene that post-translationally promotes MYC expression. Repression of this enhancer inhibited growth by 2-to-3-fold. Finally, the promoter of DOCK2 was amongst the highest negatively selected elements. DOCK2 is a guanine nucleotide exchange factor (GEF) that is expressed primarily in hematopoietic lineages and promotes actin polymerization. Repression of the DOCK2 promoter suppressed expression of DOCK2 by 10-to-100-fold and inhibited growth by 2-to-5-fold in multiple cell lines. Repressing the DOCK2 promoter had no effect on the nearby pan-essential gene SPDL1. To orthogonally confirm these findings, we created a THP-6-DOCK2-FKBPF36V degron cell line in which the C-terminus of DOCK2 is tagged with FKBPF36V. Western blot confirmed knockdown of DOCK2 upon treatment of these cells with dTag-V1. DOCK2 knockdown reduced total filamentous actin by 2-fold and slowed proliferation by 2-to-4-fold when compared with wild-type control. These findings suggest for the first time that cytoskeletal remodeling might drive cell intrinsic ALL proliferation. Taken together, our studies applied a novel approach to define the downstream “regulome” of ETS1 in T-ALL. We revealed a previously uncharacterized network of novel cell-specific non-coding regulatory elements and genes that are important for T-ALL viability.

Abstract We sought to identify mechanisms that activate MYC expression in germinal center B cell-(GCB) diffuse large B-cell lymphomas (DLBCL) in the absence of MYC rearrangement. High-throughput CRISPR-interference screens in MYC-rearranged (n=4) and non-MYC-rearranged (n=2) GCB-DLBCL cell lines revealed a distal enhancer complex 436 kb downstream of MYC, GME-1, that was uniquely essential in non-rearranged (MYC-intact) cell lines. CRISPR-interference studies confirmed growth inhibition and MYC downregulation upon GME-1 repression in 4 DLBCL cell lines. ChIP-Seq showed that GME-1 subunits were bound by OCT2, OCA-B, and MEF2B, which are known to activate germinal center-specific enhancers, and knockdown of the corresponding genes reduced MYC expression. GME-1 subunits showed GCB-specific acetylation and accessibility across normal human B cell populations in public chromatin datasets. The mouse ortholog of GME-1 showed germinal center B cell-specific accessibility. GME-1 was strongly acetylated in MYC-intact but not MYC-rearranged GCB-DLBCL cell lines and patient biopsies. Hi-C revealed significant looping of GME-1 to the MYC promoter in MYC protein+, MYC-intact DLBCL biopsies (n=2), but not MYC-rearranged DLBCL (n=2). We used sequence conservation and chromatin accessibility data to define four GME-1 subunits (195A/B and196A/B), which were cloned into luciferase enhancer reporter constructs and compared to two subunits of the BCL6 distal super-enhancer complex that had previously been identified as regulated by the OCT2/OCA-B/MEF2B complex. GME-1 subunit 195B was the only strongly transcription-activating element based on luciferase assays across three MYC-intact cells lines, where it showed activity comparable to BCL6 enhancers. In contrast, GME-1 enhancers were minimally active in reporter assays performed in two MYC-rearranged GCB-DLBCL cell lines. Deletion of a conserved OCT2 binding site or knockdown of the POU2F2 and POU2AF1 genes reduced activity of GME-1. Thus GME-1 trans-regulation is divergent from that of previously characterized GCB enhancers, despite shared dependency on the OCT2 ternary complex. Whole-genome sequencing (WGS) revealed that the GME-1 dependent cell line SUDHL5 bears a 176bp tandem duplication within GME-1 195B. This altered enhancer showed stronger luciferase reporter activity across three MYC-intact cell lines. Published WGS datasets showed isolated genomic tandem copy gains of GME-1 in 2/49 biopsies from recurrent/refractory DLBCL and 1/93 unselected DLBCL. In a cohort of 18 DLBCL with the high-risk DZSig gene expression signature, GME-1 amplification was seen in two biopsies with concurrent deletion of the PVT1 promoter (PVT1-P) and no inter-chromosomal MYC locus rearrangement. CRISPRi showed that PVT1-P repression reduced growth and MYC expression in GME-1-dependent DLBCL, but not in cell lines where rearrangements removed PVT-P from its position in cis with the MYC gene. Our findings identify GME-1 as an activator of MYC expression and target of rare but recurrent genomic copy gains in high-risk DLBCL. Citation Format: Aishwarya Gurumurthy, Ashwin R. Iyer, Shih-Chun A. Chu, Marcin P. Cieslik, Russell J.H. Ryan. Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR04.

In T-cell acute lymphoblastic leukemia (T-ALL), Notch variants are the most common oncogenic mutations, but clinical trials showed excessive toxicity of pan-Notch inhibitors. In response, we refocused to ETS1, which we and others previously showed is the top transcription factor that most frequently co-binds Notch-occupied elements in T-ALL. Here, we performed an unbiased genome-wide CRISPR-interference screen of the strongest ETS1-dependent enhancers. The #2 ranked element was the Notch-MYC enhancer (N-Me). The #1 ranked element was a +140kb Notch-bound enhancer interacting with the MYB promoter that we named the “ETS-MYB enhancer” or E-Me. MYB is an oncogene across all T-ALL subgroups. Chromatin profiling showed that the E-Me is highly active and accessible in hematopoietic stem cells (HSCs) and then silenced during differentiation. In contrast, the E-Me is highly active in Notch-type T-ALL. Therefore, we hypothesized that the E-Me is a HSC enhancer that is reactivated in T-ALL cells to drive MYB expression and promote population cell growth. To test this, we first determined the physiological role of the E-Me in hematopoietic cells using a novel E-Me conditional knockout mouse. E-Me deletion reduced Myb expression in HSCs 2-fold and increased absolute numbers 2.1-fold, whereas other hematopoietic populations were minimally affected. E-Me-deficient HSCs were defective as they reconstituted poorly in serial competitive bone marrow transplants and were depleted 6.2-fold in aged mice. Lastly, germline E-Me inactivation had no long-term effects on mouse weight or survival. These data suggest that the E-Me has limited function in committed T-cell progenitors and normal physiology but is important for HSC long-term self-renewal. We next wanted to examine E-Me function in murine and human T-ALL leukemogenesis. To do this, we first generated Notch-induced and Lmo2-induced T-ALL mouse models. E-Me deletion during initiation or maintenance reduced blast counts 38-to-105 fold and significantly prolonged survival. Next, we transduced E-Me sgRNAs into human T-ALL cell lines, which suppressed MYB expression 3.3-to-5.3-fold and inhibited cell proliferation 17-to-113-fold. These results indicate that the E-Me has major importance in murine and human leukemic cell growth and MYB induction. Following this, we wanted to understand how ETS1 induces E-Me activity. We first performed HOMER analysis of the E-Me and identified a single conserved ETS1-binding motif. Mutating this site decreased pulldown of ETS1 but no other transcription factor in reverse ChIP mass spectrometry and abrogated E-Me activity in reporter assays. In contrast, Notch inhibitors had no effect. Next, we generated a mouse model with mutation of the ETS1 site. Sequencing of ETS1 ChIP pulldowns confirmed the inability of ETS1 to bind the mutated motif. These mutant mice showed no significant alterations during steady state thymopoiesis but had impaired T-cell regeneration after sublethal irradiation. Dual reverse ChIP and co-IP mass spectrometry screens in a T-ALL cell line identified cBAF, a chromatin remodeling complex, as a top ranked ETS1 cofactor that is recruited to the E-Me. To confirm this, we genetically degraded ETS1 and found 2.5-fold reduced cBAF occupancy, 3.2-fold reduced H3K27ac signals, and 4.9-fold reduced ATAC-seq signals at the E-Me. ETS1 deprivation also reduced occupancy of Notch-associated transcriptional regulators. Lastly, AU-15330, a PROTAC degrader of cBAF, impaired E-Me H3K27ac signals 2.1-11.1-fold and reduced MYB protein levels 2.3-9.7-fold. These data suggest that ETS1 recruits cBAF after transformation to promote chromatin accessibility at the E-Me. There is an unmet need to identify the most important oncogenic enhancers and find ways to safely eject transcription factors bound to these elements as potential therapies. In addressing this, our unbiased screen revealed the top importance of a stem cell enhancer, the E-Me, which was ranked higher than the N-Me. We also suggest ways to inactivate the E-Me with cBAF and ETS1 degraders, which mouse studies predict would be safer than pan-Notch or pan-MYB inhibition. Finally, the literature provides ample examples of Notch having a central role in T-ALL. In contrast, we identify a top-ranked oncogenic enhancer that is independent of Notch but requires ETS1 to remodel chromatin to enable transcription factor complex assembly and function.

Introduction:The relationship between vitamin D receptor (VDR) polymorphisms and cardiovascular disease (CVD) is unclear.This study explores the correlation between VDR genotypes, plasma concentrations of vitamin D metabolites, and CVD occurrence and metabolic disorders.Method: Fifty-eight patients with CVD were included.ApaI rs7975232 and TaqI rs731236 were genotyped using PCR-RFLP method.Circulating 25-hydroxyvitamin-D2, 25-hydroxyvitamin-D3, and 3-epi-25-hydroxyvitamin D3 were measured by a validated UPLC-MS/MS method.Results: ApaI polymorphism frequencies were significantly (p = 0.01) associated with hypertension cases (GG 71%, GT 48%, TT 93%).Patients with ApaI-GG genotype had significantly (p < 0.01) higher plasma levels of 25(OH)D3 and 3-epi-25(OH)D3 compared with ApaI-GT carriers.Backward stepwise regression shows that hypertension can be predicted by BMI (OR=2.11,95%CI=0.08-1.41,p=0.03),HbA1C[%] (OR=32.39,95%CI= 0.17-6.79,p= 0.04), while ApaI-GT was protective (OR=0.05,95%CI=-5.92-(-0.02),p= 0.03).For TaqI genotypes, significant differences between the values were found in BMI (p = 0.04), and obesity incidence (CC 22%, TC 50%, TT 12%; p<0.01) and hypercholesterolemia (CC 67%, TC 45%, TT 69%; p=0.05).3-epi-25(OH) D3 levels were significantly lower in TaqI-TC genotype compared with TaqI-CC genotype (p=0.03).Discussion: ApaI-GT genotype can be protective against hypertension.Taq-TC and Taq-TT genotypes were associated with a higher risk of obesity and hypercholesterolemia, respectively.ApaI-GT and Taq-CC genotype were associated with higher 25-hydroxyvitamin-D levels.Further research is required to confirm these outcomes and to investigate their potential mechanisms.

Abstract Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6–RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6–RUNX1+/− like B-ALL “signature” genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6–RUNX1+/− like B-ALL. Significance: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1

High expression of MYC and its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding gene PVT1 are enriched in MYC -intact cases. To identify genomic drivers of MYC activation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in the MYC locus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements between MYC and immunoglobulin (Ig) loci. Rearrangements between MYC and non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within the BCL6 super-enhancer ( BCL6 -SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrent MYC::BCL6 -SE rearrangement. In contrast, GCB-DLBCL cell lines without MYC rearrangement were highly dependent on a previously uncharacterized 3' enhancer within the MYC locus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that the PVT1 promoter limits MYC activation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3' rearrangements that remove PVT1 from its position in cis with the rearranged MYC gene. Key points:CRISPR-interference screens identify a conserved germinal center B cell MYC enhancer that is essential for GCB-DLBCL lacking MYC rearrangements. Functional profiling of MYC partner loci reveals principles of MYC enhancer-hijacking activation by non-immunoglobulin rearrangements.

Strong expression of MYC and MYC-regulated genes defines the molecular high-grade / double-hit signature subgroup of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) which shows poor clinical outcomes with current therapy. About half of such cases show large-scale chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, but there is little understanding of the specific regulatory elements that activate MYC in absence of a rearrangement, or in the setting of non-immunoglobulin MYC rearrangement partners (RPs). We used high-throughput CRISPR-interference functional profiling (CRISPRi) to pinpoint cis-regulatory element dependencies in MYC-intact and MYC-rearranged lymphoma cell lines at the level of individual nucleosome-free regions (NFRs). Custom sgRNA libraries targeted 233 individual NFRs (median 42 sgRNA / NFR after stringent quality filtering) across the MYC topological domain (TAD) and MYC RP loci in six GCB-DLBCL cell lines and two comparator mantle cell lymphoma (MCL) cell lines. Promoter-targeting sgRNAs against 587 sequence-specific transcription factor (TF) genes and 65 essential gene controls allowed for interrogation of trans-factor dependencies in the same assay. CRISPRi profiling in one GCB-DLBCL and one MCL cell line with intra-chromosomal rearrangements of chromosome 8 showed strong, unique dependencies on enhancers within the cell-line-specific RPs, supporting an "enhancer-hijacking" function of these rearrangements. Three profiled cell lines showed structurally distinct variants of a recurrent t(3;8)(q27;q24) rearrangement that links the MYC gene to the distal super-enhancer region 150-240 kB upstream of BCL6. Although the distal BCL6 super-enhancers have been shown to sustain the essential BCL6 oncogene itself, we surprisingly found only weak dependency on the distal BCL6 enhancers for the three GCB-DLBCL cell lines lacking t(3;8) rearrangements. In contrast, all three GCB-DLBCL cell lines with t(3;8) rearrangements showed strong dependency on specific NFRs within the distal BCL6 super-enhancer. Validation assays showed that concurrent targeting of two BCL6 enhancer NFRs lead to even greater growth suppression and loss of MYC expression in t(3;8)-bearing cell lines. In both GCB-DLBCL cell lines lacking MYC rearrangements, we identified a strong, novel dependency on a specific enhancer ("GCB-ME1") in the 3' portion of the MYC TAD. This enhancer was acetylated in MYC-intact GCB-DLBCL patient samples and normal centroblasts, but not in MCL or MYC-rearranged DLBCL samples. We confirmed that sustained MYC expression in MYC-intact cell lines requires activity of GCB-ME1. GCB-ME1 showed little or no essentiality in the four MYC-rearranged DLBCL cell lines or in the MYC-intact MCL cell line SP-49, which is dependent on previously identified Notch/RBPJ-regulated 5' MYC enhancers (Ryan et al., 2017). ChIP-Seq showed that both GCB-ME1 and all of the most strongly essential BCL6 enhancers in t(3;8) cell lines were strongly bound by a triad of TFs, MEF2B, POU2F2, and POU2AF1, that were previously implicated in BCL6 activation. These genes were required to sustain MYC expression in both MYC-intact and t(3;8) GCB-DLBCL, and were highly essential in those cell lines, but POU2F2 and MEF2B were not essential in some cell lines with alternate MYC rearrangements. Our data show that DLBCL is highly sensitive to selective disruption of discrete MYC-activating enhancers. Transcriptional regulatory factor dependencies in DLBCL may be shaped by the requirement to sustain MYC activation via the native GCB-ME1 enhancer or alternately via enhancers in specific RP loci, a finding with implications for therapeutic targeting of transcriptional regulatory mechanisms in DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phospholipase D4 (PLD4), a single-pass transmembrane glycoprotein, is among the most highly upregulated genes in murine kidneys subjected to chronic progressive fibrosis, but the function of PLD4 in this process is unknown. Here, we found PLD4 to be overexpressed in the proximal and distal tubular epithelial cells of murine and human kidneys after fibrosis. Genetic silencing of PLD4, either globally or conditionally in proximal tubular epithelial cells, protected mice from the development of fibrosis. Mechanistically, global knockout of PLD4 modulated innate and adaptive immune responses and attenuated the upregulation of the TGF-β signaling pathway and α1-antitrypsin protein (a serine protease inhibitor) expression and downregulation of neutrophil elastase (NE) expression induced by obstructive injury. In vitro, treatment with NE attenuated TGF-β-induced accumulation of fibrotic markers. Furthermore, therapeutic targeting of PLD4 using specific siRNA protected mice from folic acid-induced kidney fibrosis and inhibited the increase in TGF-β signaling, decrease in NE expression, and upregulation of mitogen-activated protein kinase signaling. Immunoprecipitation/mass spectrometry and coimmunoprecipitation experiments confirmed that PLD4 binds three proteins that interact with neurotrophic receptor tyrosine kinase 1, a receptor also known as TrkA that upregulates mitogen-activated protein kinase. PLD4 inhibition also prevented the folic acid-induced upregulation of this receptor in mouse kidneys. These results suggest inhibition of PLD4 as a novel therapeutic strategy to activate protease-mediated degradation of extracellular matrix and reverse fibrosis.

Secreted modular calcium-binding protein 2 (SMOC2) belongs to the secreted protein acidic and rich in cysteine (SPARC) family of matricellular proteins whose members are known to modulate cell-matrix interactions. We report that SMOC2 is upregulated in the kidney tubular epithelial cells of mice and humans following fibrosis. Using genetically manipulated mice with SMOC2 overexpression or knockdown, we show that SMOC2 is critically involved in the progression of kidney fibrosis. Mechanistically, we found that SMOC2 activates a fibroblast-to-myofibroblast transition (FMT) to stimulate stress fiber formation, proliferation, migration, and extracellular matrix production. Furthermore, we demonstrate that targeting SMOC2 by siRNA results in attenuation of TGFβ1-mediated FMT in vitro and an amelioration of kidney fibrosis in mice. These findings implicate that SMOC2 is a key signaling molecule in the pathological secretome of a damaged kidney and targeting SMOC2 offers a therapeutic strategy for inhibiting FMT-mediated kidney fibrosis - an unmet medical need.