HTRA1 disaggregates α-synuclein amyloid fibrils and converts them into non-toxic and seeding incompetent species.

Parkinson's disease (PD) is closely linked to α-synuclein (α-syn) misfolding and accumulation in Lewy bodies. The PDZ serine protease HTRA1 degrades fibrillar tau, which is associated with Alzheimer's disease, and inactivating mutations to mitochondrial HTRA2 are implicated in PD. Here, we report that HTRA1 inhibits aggregation of α-syn as well as FUS and TDP-43, which are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The protease domain of HTRA1 is necessary and sufficient for inhibiting aggregation, yet this activity is proteolytically-independent. Further, HTRA1 disaggregates preformed α-syn fibrils, rendering them incapable of seeding aggregation of endogenous α-syn, while reducing HTRA1 expression promotes α-syn seeding. HTRA1 remodels α-syn fibrils by targeting the NAC domain, the key domain catalyzing α-syn amyloidogenesis. Finally, HTRA1 detoxifies α-syn fibrils and prevents formation of hyperphosphorylated α-syn accumulations in primary neurons. Our findings suggest that HTRA1 may be a therapeutic target for a range of neurodegenerative disorders.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple Myeloma (MM) is an incurable plasma cell malignancy, that despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival and recently the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (~20%), there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in two non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death and interestingly we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples, that 5-azacytidine enhances the response to venetoclax ex-vivo, across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identifies 5-azacytidine and venetoclax as an effective treatment combination and this could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Acquired Multidrug Resistance in AML Is Caused by Low Apoptotic Priming in Relapsed Myeloblasts.

In many cancers, mortality is associated with the emergence of relapse with multidrug resistance (MDR). Thus far, the investigation of cancer relapse mechanisms has largely focused on acquired genetic mutations. Using acute myeloid leukemia (AML) patient-derived xenografts (PDX), we systematically elucidated a basis of MDR and identified drug sensitivity in relapsed AML. We derived pharmacologic sensitivity for 22 AML PDX models using dynamic BH3 profiling (DBP), together with genomics and transcriptomics. Using in vivo acquired resistant PDXs, we found that resistance to unrelated, narrowly targeted agents in distinct PDXs was accompanied by broad resistance to drugs with disparate mechanisms. Moreover, baseline mitochondrial apoptotic priming was consistently reduced regardless of the class of drug-inducing selection. By applying DBP, we identified drugs showing effective in vivo activity in resistant models. This study implies evasion of apoptosis drives drug resistance and demonstrates the feasibility of the DBP approach to identify active drugs for patients with relapsed AML. SIGNIFICANCE: Acquired resistance to targeted therapy remains challenging in AML. We found that reduction in mitochondrial priming and common transcriptomic signatures was a conserved mechanism of acquired resistance across different drug classes in vivo. Drugs active in vivo can be identified even in the multidrug resistant state by DBP.

Prophylactic maintenance with venetoclax/azacitidine after reduced-intensity conditioning allogeneic transplant for high-risk MDS and AML.

ABSTRACT: We conducted a phase 1 trial assessing safety and efficacy of prophylactic maintenance therapy with venetoclax and azacitidine (Ven/Aza) for patients with high-risk myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) undergoing reduced intensity allogeneic stem cell transplantation (allo-SCT) after Ven and fludarabine/busulfan conditioning (Ven/FluBu2 allo-SCT) with tacrolimus and methotrexate as graft-versus-host disease (GVHD) prophylaxis. Among 27 patients who underwent Ven/FluBu2 allo-SCT (55.6% with prior Ven exposure, and 96% with positive molecular measurable residual disease), 22 received maintenance therapy with Aza 36 mg/m2 intravenously on days 1 to 5, and Ven 400 mg by mouth on days 1 to 14 per assigned dose schedule/level (42-day cycles × 8, or 28-day cycles × 12). During maintenance, the most common grade 3-4 adverse events were leukopenia, neutropenia, and thrombocytopenia, which were transient and manageable. Infections were uncommon (n = 4, all grade 1-2). The 1-year and 2-year moderate/severe chronic GVHD rates were 4% (95% confidence interval [CI], 0.3%-18%) and 22% (95% CI, 9%-40%), respectively. After a median follow-up of 25 months among survivors, the median overall survival (OS) was not reached. Among the 22 patients who received Ven/Aza maintenance, the 2-year OS, progression-free survival, nonrelapse mortality, and cumulative incidence of relapse rates were 67% (95% CI, 43%-83%), 59% (95% CI, 36%-76%), 0%, and 41% (95% CI, 20%-61%), respectively. Immune monitoring demonstrated no significant impact on T-cell expansion but identified reduced B-cell expansion compared with controls. This study demonstrates prophylactic Ven/Aza maintenance can be safely administered for patients with high-risk MDS/AML, but a randomized study is required to properly assess any potential benefit. This trial was registered at www.clinicaltrials.gov as #NCT03613532.

Amyloidogenic propensity of self-assembling peptides and their adjuvant potential for use as DNA vaccines.

De novo designed peptides that self-assemble into cross-ß rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-ß peptide KFE8, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE8 fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-ß-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. STATEMENT OF SIGNIFICANCE: Biomaterials comprised of self-assembling peptides hold great promise for the development of new vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-ß rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.

Sequential apoptotic and multiplexed proteomic evaluation of single cancer cells.

A potential cause of cancer relapse is pretreatment chemoresistant subpopulations. Identifying targetable features of subpopulations that are poorly primed for therapy-induced cell death may improve cancer therapy. Here, we develop and validate real-time BH3 profiling, a live and functional single-cell measurement of pretreatment apoptotic sensitivity that occurs upstream of apoptotic protease activation. On the same single cells, we perform cyclic immunofluorescence, which enables multiplexed immunofluorescence of more than 30 proteins on the same cell. Using cultured cells and rapid ex vivo cultures of colon cancer patient-derived xenograft (PDX) models, we identify Bak as a univariate correlate of apoptotic priming, find that poorly primed subpopulations can correspond to specific stages of the cell cycle, and, in some PDX models, identify increased expression of Bcl-XL, Mcl-1, or Her2 in subpopulations that are poorly primed for apoptosis. Last, we generate and validate mathematical models of single-cell priming that describe how targetable proteins contribute to apoptotic priming.

Probing the drivers of Staphylococcus aureus biofilm protein amyloidogenesis and disrupting biofilms with engineered protein disaggregases.

Phenol-soluble modulins (PSMs) are the primary proteinaceous component of Staphylococcus aureus biofilms. Residence in the protective environment of biofilms allows bacteria to rapidly evolve and acquire antimicrobial resistance, which can lead to persistent infections such as those caused by methicillin-resistant S. aureus (MRSA). In their soluble form, PSMs hinder the immune response of the host and can increase the virulence potential of MRSA. PSMs also self-assemble into insoluble functional amyloids that contribute to the structural scaffold of biofilms. The specific roles of PSM peptides in biofilms remain poorly understood. Here, we report the development of a genetically tractable yeast model system for studying the properties of PSMα peptides. Expression of PSMα peptides in yeast drives the formation of toxic insoluble aggregates that adopt vesicle-like structures. Using this system, we probed the molecular drivers of PSMα aggregation to delineate key similarities and differences among the PSMs and identified a crucial residue that drives PSM features. Biofilms are a major public health threat; thus, biofilm disruption is a key goal. To solubilize aggregates comprised of a diverse range of amyloid and amyloid-like species, we have developed engineered variants of Hsp104, a hexameric AAA+ protein disaggregase from yeast. Here, we demonstrate that potentiated Hsp104 variants counter the toxicity and aggregation of PSMα peptides. Further, we demonstrate that a potentiated Hsp104 variant can drive the disassembly of preformed S. aureus biofilms. We suggest that this new yeast model can be a powerful platform for screening for agents that disrupt PSM aggregation and that Hsp104 disaggregases could be a promising tool for the safe enzymatic disruption of biofilms. IMPORTANCE Biofilms are complex mixtures secreted by bacteria that form a material in which the bacteria can become embedded. This process transforms the properties of the bacteria, and they become more resistant to removal, which can give rise to multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we study phenol-soluble modulins (PSMs), which are amyloidogenic proteins secreted by S. aureus, that become incorporated into biofilms. Biofilms are challenging to study, so we have developed a new genetically tractable yeast model to study the PSMs. We used our system to learn about several key features of the PSMs. We also demonstrate that variants of an amyloid disaggregase, Hsp104, can disrupt the PSMs and, more importantly, dissolve preformed S. aureus biofilms. We propose that our system can be a powerful screening tool and that Hsp104 disaggregases may be a new avenue to explore for biofilm disruption agents.

Quantifying requirements for mitochondrial apoptosis in CAR T killing of cancer cells.

Chimeric antigen receptor (CAR) T cell therapy is an FDA-approved treatment for several hematologic malignancies, yet not all patients respond to this treatment. While some resistance mechanisms have been identified, cell death pathways in target cancer cells remain underexplored. Impairing mitochondrial apoptosis via knockout of Bak and Bax, forced Bcl-2 and Bcl-XL expression, or caspase inhibition protected several tumor models from CAR T killing. However, impairing mitochondrial apoptosis in two liquid tumor cell lines did not protect target cells from CAR T killing. We found that whether a cell was Type I or Type II in response to death ligands explained the divergence of these results, so that mitochondrial apoptosis was dispensable for CART killing of cells that were Type I but not Type II. This suggests that the apoptotic signaling induced by CAR T cells bears important similarities to that induced by drugs. Combinations of drug and CAR T therapies will therefore require tailoring to the specific cell death pathways activated by CAR T cells in different types of cancer cells.

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction.

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion.

Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN, the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1, individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 ­ and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1-deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.

Lisaftoclax (APG-2575) Is a Novel BCL-2 Inhibitor with Robust Antitumor Activity in Preclinical Models of Hematologic Malignancy.

PURPOSE: Development of B-cell lymphoma 2 (BCL-2)-specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein-protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). EXPERIMENTAL DESIGN: Computational modeling was used to design "lead" compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. RESULTS: Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. CONCLUSIONS: These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2-selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.

Augmenting NK cell-based immunotherapy by targeting mitochondrial apoptosis.

Interest in harnessing natural killer (NK) cells for cancer immunotherapy is rapidly growing. However, efficacy of NK cell-based immunotherapy remains limited in most trials. Strategies to augment the killing efficacy of NK cells are thus much needed. In the current study, we found that mitochondrial apoptosis (mtApoptosis) pathway is essential for efficient NK killing, especially at physiologically relevant effector-to-target ratios. Furthermore, NK cells can prime cancer cells for mtApoptosis and mitochondrial priming status affects cancer-cell susceptibility to NK-mediated killing. Interestingly, pre-activating NK cells confers on them resistance to BH3 mimetics. Combining BH3 mimetics with NK cells synergistically kills cancer cells in vitro and suppresses tumor growth in vivo. The ideal BH3 mimetic to use in such an approach can be predicted by BH3 profiling. We herein report a rational and precision strategy to augment NK-based immunotherapy, which may be adaptable to T cell-based immunotherapies as well.

Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia.

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML.

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275.

Adding venetoclax to fludarabine/busulfan RIC transplant for high-risk MDS and AML is feasible, safe, and active.

Adding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day -8 for 6-7 doses) in combination with fludarabine 30 mg/m2 per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day -5 to day -2 (FluBu2). Transplant related-toxicity was evaluated from the first venetoclax dose on day -8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.gov as #NCT03613532.

Mitochondrial apoptotic priming is a key determinant of cell fate upon p53 restoration.

Reactivation of p53 in established tumors typically results in one of two cell fates, cell cycle arrest or apoptosis, but it remains unclear how this cell fate is determined. We hypothesized that high mitochondrial priming prior to p53 reactivation would lead to apoptosis, while low priming would lead to survival and cell cycle arrest. Using a panel of Kras-driven, p53 restorable cell lines derived from genetically engineered mouse models of lung adenocarcinoma and sarcoma (both of which undergo cell cycle arrest upon p53 restoration), as well as lymphoma (which instead undergo apoptosis), we show that the level of mitochondrial apoptotic priming is a critical determinant of p53 reactivation outcome. Cells with high initial priming (e.g., lymphomas) lacked sufficient reserve antiapoptotic capacity and underwent apoptosis after p53 restoration. Forced BCL-2 or BCL-XL expression reduced priming and resulted in survival and cell cycle arrest. Cells with low initial priming (e.g., lung adenocarcinoma and sarcoma) survived and proceeded to arrest in the cell cycle. When primed by inhibition of their antiapoptotic proteins using genetic (BCL-2 or BCL-XL deletion or BAD overexpression) or pharmacologic (navitoclax) means, apoptosis resulted upon p53 restoration in vitro and in vivo. These data demonstrate that mitochondrial apoptotic priming is a key determining factor of cell fate upon p53 activation. Moreover, it is possible to enforce apoptotic cell fate following p53 activation in less primed cells using p53-independent drugs that increase apoptotic priming, including BH3 mimetic drugs.

Drivers of Hsp104 potentiation revealed by scanning mutagenesis of the middle domain.

Hsp104, a yeast protein disaggregase, can be potentiated via numerous missense mutations at disparate locations throughout the coiled-coil middle domain (MD). Potentiated Hsp104 variants can counter the toxicity and misfolding of TDP-43, FUS, and α-synuclein, proteins which are implicated in neurodegenerative disorders. However, potentiated MD variants typically exhibit off-target toxicity. Further, it has remained confounding how numerous degenerate mutations confer potentiation, hampering engineering of therapeutic Hsp104 variants. Here, we sought to comprehensively define the key drivers of Hsp104 potentiation. Using scanning mutagenesis, we iteratively studied the effects of modulation at each position in the Hsp104 MD. Screening this library to identify enhanced variants reveals that missense mutations at 26% of positions in the MD yield variants that counter FUS toxicity. Modulation of the helix 2-helix 3/4 MD interface potentiates Hsp104, whereas mutations in the analogous helix 1-2 interface do not. Surprisingly, we find that there is a higher likelihood of enhancing Hsp104 activity against human disease substrates than impairing Hsp104 native function. We find that single mutations can broadly destabilize the MD structure and lead to functional potentiation, suggesting this may be a common mechanism conferring Hsp104 potentiation. Using this approach, we have demonstrated that modulation of the MD can yield engineered variants with decreased off-target effects.

Apoptotic Blocks in Primary Non-Hodgkin B Cell Lymphomas Identified by BH3 Profiling.

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.