Biology subjects

Wertheim, G.

Publications and source records attributed to Wertheim, G..

7 recordsLinked to original sources

bioRxiv2025

Pitavastatin counteracts venetoclax resistance mechanisms in acute myeloid leukemia

The BCL2 inhibitor venetoclax has therapeutic activity in several hematological malignancies. In acute myeloid leukemia (AML), venetoclax combined with hypomethylating agents is the standard of care for patients unfit for intensive chemotherapy, but intrinsic and acquired resistance are common. Loss of p53 function is strongly associated with venetoclax resistance, and adding venetoclax to 5-azacitidine provides no overall survival benefit in TP53-mutant AML. Other frequent mechanisms of venetoclax resistance in AML include FLT3 mutations, MCL-1 upregulation, and altered mitochondrial metabolism. Unfortunately, it has been challenging to develop agents that target these mechanisms directly and combinatorially. Here we report that pitavastatin, an inhibitor of HMG-CoA-reductase, promotes apoptosis and overcomes several venetoclax resistance mechanisms in human AML cells. At clinically achievable concentrations, pitavastatin treatment has potent cytotoxic activity in cells with mutations in TP53 or FLT3. The apoptotic mechanism involves p53-independent PUMA upregulation and reduced MCL-1 expression. Pitavastatin also suppresses mitochondrial gene expression and oxidative metabolism. The pro-apoptotic actions of pitavastatin depend on depletion of geranylgeranyl pyrophosphate (GGPP) and can be recapitulated by inhibiting GGPP synthase or geranylgeranyltransferase-1 enzymes. These results provide a mechanistic rationale for adding pitavastatin to AML regimens to prevent or overcome venetoclax resistance.

cancer biology↗

bioRxiv2025

Selective Sensitivity of Ph-like B-ALL to BRG1 Inhibition Reveals a Novel Targeted Therapy Strategy

Despite therapeutic advances, high-risk subtypes of B-cell acute lymphoblastic leukemia (B-ALL) such as Philadelphia chromosome-like (Ph-like) and KMT2A-rearranged (KMT2A-R) remain a formidable clinical challenge. BRG1 (gene name SMARCA4), the ATPase subunit of the SWI/SNF chromatin-remodeling complex, has been extensively studied in solid tumors, where inactivating mutations are linked to aggressive disease and poor prognosis. Although BRG1 is known to be essential for early B cell development, its role in B-ALL remains poorly understood. Therefore, we investigated the therapeutic relevance of BRG1 in high-risk B-ALL. Meta-analysis of gene expression data revealed that BRG1-inactivating mutations are exceedingly rare (0.35%) in B-ALL, suggesting that intact BRG1 function may be critical for leukemogenesis. Subtype-specific analyses revealed that elevated BRG1 expression is associated with significantly shorter overall survival in children with Ph-like B-ALL, while the opposite trend was observed in KMT2A-R B-ALL. We confirmed higher BRG1 expressions in Ph-like compared to KMT2A-R B-ALL via gene expression analysis, RT-PCR, and Western blotting. The pharmacologic inhibition of BRG1 using two selective inhibitors, BRM014 and FHD-286, revealed marked sensitivity in Ph-like B-ALL cell lines, whereas KMT2A-R B-ALL was resistant. Mechanistically, we found that BRG1 inhibition results in cell cycle arrest via downregulation of cell cycle regulators (CCND3, CDK4, CDK6, E2F1, and MYC) and upregulation of the cell cycle inhibitor CDKN1B (p27). Importantly, treatment with FHD-286 significantly prolonged the survival of NSG mice engrafted with Ph-like B-ALL cells. Taken together, these findings establish BRG1 as a critical, subtype-specific dependency in Ph-like B-ALL and demonstrate that its pharmacologic inhibition effectively suppresses leukemic cell proliferation through induction of cell cycle arrest. The pronounced in vitro sensitivity and improved in vivo survival upon BRG1 inhibition provide compelling preclinical evidence for its therapeutic targeting. These results support the advancement of BRG1-directed strategies as a viable treatment approach for patients with Ph-like B-ALL, with the potential to improve outcomes in this high-risk population. HighlightsO_LIHigher levels of BRG1 correlate to poor clinical outcomes in Ph-like but KMT2A-R B-ALL C_LIO_LIInhibition of BRG1 induces cell cycle arrest in Ph-like cells in vitro and extends the survival of mice in pre-clinical in vivo studies C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/661805v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1b0a6cforg.highwire.dtl.DTLVardef@977257org.highwire.dtl.DTLVardef@d3bf7forg.highwire.dtl.DTLVardef@1c12d52_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Chemoresistance of TP53 mutant AML requires the mevalonate byproduct, GGPP, for regulation of ROS and induction of a mitochondria stress response

Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene, TP53 (TP53mut AML), is fatal with a median survival of only 6 months. RNA sequencing on purified AML patient samples show TP53mut AML has higher expression of mevalonate pathway genes. We retrospectively identified a survival benefit in TP53mut AML patients who received chemotherapy concurrently with a statin, which inhibits the mevalonate pathway. Mechanistically, TP53mut AML resistance to standard AML chemotherapy, cytarabine (AraC), correlates with increased mevalonate pathway activity and a mitochondria stress response with increased mitochondria mass and oxidative phosphorylation. Pretreatment with a statin reverses these effects and chemosensitizes TP53mut AML cell lines and primary samples in vitro and in vivo. Mitochondria-dependent chemoresistance requires the geranylgeranyl pyrophosphate (GGPP) branch of the mevalonate pathway and novel GGPP-dependent synthesis of glutathione to manage AraC-induced reactive oxygen species (ROS). Overall, we show that the mevalonate pathway is a novel therapeutic target in TP53mut AML. SignificanceChemotherapy-persisting TP53mut AML cells induce a mitochondria stress response that requires mevalonate byproduct, GGPP, through its novel role in glutathione synthesis and regulation of mitochondria metabolism. We provide insight into prior failures of the statin family of mevalonate pathway inhibitors in AML. We identify clinical settings and strategies to successfully target the mevalonate pathway, particularly to address the unmet need of TP53mut AML.

cancer biology↗

bioRxiv2023

Clonal evolution mediates Menin-inhibitor resistance in KMT2A-rearranged leukemias.

Inhibitors of the Menin-KMT2A interaction are promising agents for the treatment of KMT2A-rearranged (KMT2A-r) leukemias. We evaluated Menin inhibition in patient derived xenografts of KMT2A-r leukemias with high-risk features. Three AMLs with high-risk fusion partners (MLLT10, MLLT4) and two infant ALL samples were sensitive to Menin inhibition. We also evaluated serial samples from two patients with multiply relapsed ALL. We found that highly pretreated KMT2A-AFF1 ALL samples were much less sensitive compared to cells obtained earlier in the same patients disease course. Since none of the patients had been treated with a Menin inhibitor, resistance in these highly pretreated samples was acquired in the absence to Menin inhibitor exposure. Transcriptomic analysis documented sustained on-target efficacy towards the canonical targets in the Menin-inhibitor in resistant cells. Targeted genomic analysis documented the emergence of multiple co-mutations, including RAS pathway and TP53 mutations, although neither was sufficient to induce Menin-inhibitor resistance in vitro. Downregulation of KMT3D may account for resistance in one patients; inactivation of KMT2C/D had previously been reported to result in Menin inhibitor resistance. Future studies will need to clarify more broadly which genomic/epigenomic alterations drive upfront resistance. Regardless of mechanism, our data supports using Menin-inhibitors upfront or in early lines of therapy before substantial genomic or epigenomic evolution has occurred.

cancer biology↗

bioRxiv2023

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease

Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.

immunology↗

bioRxiv2022

Pharmacologic Inhibition of DYRK1A Results in MYC Hyperactivation and ERK Hyperphosphorylation rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.

cancer biology↗

bioRxiv2021

Molecular evolution of classic Hodgkin lymphoma revealed through whole genome sequencing of Hodgkin and Reed Sternberg cells

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells within a classic Hodgkin lymphoma (cHL) biopsy limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Here we leveraged this method to report the first whole genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events prior to the clinical diagnosis of cHL. We identified alterations in driver genes not previously described in cHL, a high activity of the APOBEC mutational signature, and the presence complex structural variants including chromothripsis. We found that the high ploidy observed in cHL is often acquired through multiple, independent large chromosomal gain events including whole genome duplication. The first of these likely occurs several years prior to the diagnosis of cHL, and the last gains typically occur very close to the time of diagnosis. Evolutionary timing analyses revealed that driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID driven mutagenesis usually preceded large chromosomal gains. The study provides the first temporal reconstruction of cHL pathogenesis and suggests a relatively long time course between the first pathogenic event and the clinical diagnosis.

cancer biology↗