Clinical and genomic landscape of RAS mutations in gynecologic cancers.

BACKGROUND: We aimed to describe RAS mutations in gynecologic cancers as they relate to clinicopathologic and genomic features, survival, and therapeutic implications. METHODS: Gynecologic cancers with available somatic molecular profiling data at our institution between February 2010 and August 2022 were included and grouped by RAS mutation status. Overall survival was estimated by Kaplan-Meier method, and multivariable analysis was performed using Cox proportional-hazards model. RESULTS: Of 3328 gynecologic cancers, 523 (15.7%) showed any RAS mutation. Patients with RAS-mutated tumors were younger (57 vs 60 years non-mutated), had higher prevalence of endometriosis (27.3% vs 16.9%), and lower grades (grade 1/2, 43.2% vs 8.1%, all p<0.0001). Highest prevalence of KRAS mutation was in mesonephric-like endometrial (100%, n=9/9), mesonephric-like ovarian (83.3%, n=5/6), mucinous ovarian (60.4%), and low-grade serous ovarian (44.4%) cancers. After adjustment for age, cancer type, and grade, RAS mutation was associated with worse overall survival (HR=1.3, p=0.001). Specific mutations were in KRAS (13.5%), NRAS (2.0%), and HRAS (0.51%), most commonly KRAS G12D (28.4%) and G12V (26.1%). Common co-mutations were PIK3CA (30.9%), PTEN(28.8%), ARID1A (28.0%), and TP53 (27.9%), of which 64.7% were actionable. RAS+MAPK pathway-targeted therapies were administered to 62 patients with RAS-mutated cancers. While overall survival was significantly higher with therapy (8.4 years [95%CI 5.5-12.0] vs 5.5 years [95%CI 4.6-6.6], HR=0.67, p=0.031), this effect did not persist in multivariable analysis. CONCLUSION: RAS mutations in gynecologic cancers have a distinct histopathologic distribution and may impact overall survival. PIK3CA, PTEN, and ARID1A are potentially actionable co-alterations. RAS pathway-targeted therapy should be considered.

Predictors of Oncologic Outcome in Patients Receiving Phase I Investigational Therapy for Recurrent or Metastatic Cervical Cancer.

Introduction: We aimed to identify clinical, pathologic, and treatment factors that are predictive of response and survival in patients with cervical cancer referred to phase I clinical trials. Methods: Patients with cervical cancer who received at least one dose of a phase I investigational agent at our institution between 2014 and 2022 were included. The log-rank test was used to analyze differences in progression-free survival (PFS) and overall survival (OS), and multivariable regression analysis was performed. Results: We included 65 patients with a median age of 41 years (range, 20-74), 3 prior therapies (range, 1-7), and 67.7% squamous carcinoma. The rate of distant metastasis at trial entry was 84.6%. The most common molecular alterations included PIK3CA (46.5%), PD-L1+ (46.2%), EPH (30.0%), and CREBBP (23.1%); 23.1% had received a prior checkpoint inhibitor. Phase I trials were for immunotherapy (58.5%) or targeted therapy (41.5%). The rate of biomarker matching was 21.5%. For all patients, median PFS was 3.6 months (95% CI, 2.0-5.2) and OS was 9.3 months (95% CI, 7.0-10.6). Factors at study entry associated with worse survival were presence of bone metastasis (PFS 1.6 vs 4.4 months: hazard ratio [HR], 2.8; p = 0.001; OS 3.8 vs 10.0 months: HR, 3.9; p < 0.0001) and absolute lymphocyte count below 1000/µL (PFS 1.8 vs 5.2 months: HR, 2.9; p = 0.0004; OS 7.0 vs 10.6 months: HR, 3.2; p = 0.0009). Factors associated only with worse OS were absolute neutrophil count above 4700/µL, hemoglobin below 10.5 g/dL, and smoking status. Grade 3+ treatment-related adverse events were seen in 16.9% of cases. Conclusion: Bone metastasis and absolute lymphocyte count below normal range at phase I study entry portend poor survival in patients with recurrent or metastatic cervical cancer.

Inhibition of the CD47-SIRPα axis for cancer therapy: A systematic review and meta-analysis of emerging clinical data.

CD47-SIRPα interaction acts as a "don't eat me" signal and is exploited by cancer to downregulate innate and adaptive immune surveillance. There has been intense interest to develop a mechanism of blockade, and we aimed to analyze the emerging data from early clinical trials. We performed a systematic review and meta-analysis of relevant databases and conference abstracts including clinical trials using CD47 and/or SIRPα inhibitors in cancer treatment. Nonlinear mixed models were applied for comparison of response and toxicity. We retrieved 317 articles, 24 of which were eligible. These included 771 response-evaluable patients with hematologic (47.1%) and solid tumors (52.9%). Of these, 6.4% experienced complete response, 10.4% partial response, and 26.1% stable disease for a 16.7% objective response rate (ORR), 42.8% disease control rate, and 4.8-month median duration of response. ORR was significantly higher for hematologic cancers (25.3%) than solid cancers (9.1%, p=0.042). Comparing by mechanism, seven CD47 monoclonal antibodies (mAbs) and six selective SIRPα blockers were given alone or combined with checkpoint inhibitors, targeted therapy, and/or chemotherapy. In solid cancers, selective SIRPα blockade showed a higher ORR (16.2%) than anti-CD47 mAbs (2.8%, p=0.079), which was significant for combination therapies (ORR 28.3% vs 3.0%, respectively, p=0.010). Responses were seen in head and neck, colorectal, endometrial, ovarian, hepatocellular, non-small cell lung, and HER2+gastroesophageal cancers. Dose-limiting toxicity (DLT) was seen in 3.3% of patients (5.4% anti-CD47 mAbs, 1.4% selective SIRPα blockers; p=0.01). The frequency of treatment-related adverse events (TRAEs) ≥grade 3 was 18.0%, similar between the two groups (p=0.082), and mostly laboratory abnormalities. For anti-CD47 mAbs, the most common toxicities included grade 1-2 fatigue (27.2%), headache (21.0%), and anemia (20.5%). For selective SIRPα blockers, these included grade 1-2 infusion reaction (23.1%) and fatigue (15.8%). Anti-CD47 mAbs were significantly more likely than selective SIRPα blockers to cause grade 1-2 fever, chills, nausea/vomiting, headache, and anemia. In conclusion, combination therapies using selective SIRPα blockade had higher response rates in solid tumors than anti-CD47 mAb combinations. Hematologic changes were the main TRAEs, and selective SIRPα blockers seemed to have a better grade 1-2 toxicity profile. Treatment was well-tolerated with minimal DLTs.

The Next Generation of KRAS Targeting: Reasons for Excitement and Concern.

The development of selective KRASG12C inhibitors that directly inhibit KRAS, an oncogene historically thought to be "undruggable," represents a watershed moment in oncology and developmental therapeutics. Now, as KRAS-targeted therapy moves into its second phase, there is significant excitement and anticipation for durable disease control in tumor types where options remain limited, with clinical trials testing combination therapies, indirect pan-RAS/MAP kinase pathway inhibitors, and active-state RAS(on) inhibitors. However, there is also reason for caution regarding the safety and tolerability of expanded RAS inhibition. This is evidenced by the intolerability of some combination therapies with selective KRASG12C inhibitors and foreshadowed by prior failures of combination therapies in other oncogene-driven tumors. Herein, we review the landscape of and outlook for KRAS-targeted therapies. We specifically focus upon strategies to combat resistance to KRAS-targeted therapies, and discuss the possibility of off-target or unanticipated on-target effects that may limit clinical use.

Identification of KRASG12C Mutations in Circulating Tumor DNA in Patients With Cancer.

PURPOSE: KRAS is the most mutated proto-oncogene that has been identified in cancer, and treatment of patients with KRAS mutations remains an arduous challenge. Recently, KRASG12C mutation has attracted special interest because it is now considered potentially druggable with recently developed covalent small-molecule KRASG12C inhibitors. Nevertheless, to date, there have been no large-scale analyses of liquid biopsy that include testing for KRASG12C. Here, we performed a comprehensive analysis of KRASG12C mutations in multiple cancer types, as detected by circulating tumor DNA. METHODS: We conducted a 5-year retrospective review of KRASG12C mutations in patients with cancer who had undergone Guardant360 testing between July 1, 2014, and June 30, 2019; our study included treatment-naive and previously treated patients with metastatic solid tumors. RESULTS: KRASG12C mutations were identified in 2,985 of 80,911 patients (3.7%), across > 40 tumor types. KRASG12C mutations were detected most frequently in patients with nonsquamous non-small-cell lung cancer (NSCLC; 7.5%), NSCLC of all subtypes (6.9%), cancer of unknown primary (4.1%), colorectal cancer (3.5%), squamous NSCLC (2.0%), pulmonary neuroendocrine tumors (1.9%), and pancreatic ductal adenocarcinoma (1.2%) and cholangiocarcinoma (1.2%). KRASG12C mutations were predominantly clonal (clonality > 0.9%) in patients with lung adenocarcinoma, non-NSCLC, cancer of unknown primary, NSCLC, and pancreatic ductal adenocarcinoma, and patients with colorectal cancer and breast cancer had bimodal distribution of clonal and subclonal KRASG12C mutations. CONCLUSION: Our study demonstrates the feasibility of using circulating tumor DNA to identify KRASG12C mutations across solid tumors; the highest detection rate was in lung cancer, as previously reported in the literature.

Adoptive cell therapy in gynecologic cancers: A systematic review and meta-analysis.

Adoptive cell therapy (ACT) has shown promise in hematologic and solid tumors. While data supports immunogenicity of gynecologic cancers, the benefit of ACT is not yet clear. To address this question, we performed a comprehensive systematic review and meta-analysis. Eligible studies included those reporting oncologic response or toxicity data in at least one patient with any gynecologic cancer treated with ACT. Chi-square test and multivariable logistic regression were performed to identify predictors of response. We retrieved 281 articles, and 28 studies met our inclusion criteria. These comprised of 401 patients including 238 patients with gynecologic cancers (61.8% ovarian, 34.0% cervical, 2.9% endometrial, and 1.2% other). In patients with gynecologic cancers, response rates to ACT were 8.1% complete response, 18.2% partial response, and 31.4% stable disease, for an objective response rate (ORR) of 26.3%, disease control rate (DCR) of 57.6%, and median response duration of 5.5 months. Patients in studies reporting ≤1 median line of prior therapy had a higher ORR (52.9% vs. 22.6% for >1, p < 0.001), although DCR in the >1 group was still 53.2%. ORRs by ACT type were tumor infiltrating lymphocytes (TIL) 41.4%, natural killer cells 26.7%, peripheral autologous T-cells 18.4%, T-cell receptor-modified T-cells 15.4%, and chimeric antigen receptor T-cells 9.5% (p = 0.001). ORR was significantly improved with inclusion of lymphodepletion (34.8% vs. 15.4% without, p = 0.001). On multivariable analysis controlling for cancer type and lymphodepletion, TIL therapy was predictive of objective response (odds ratio 2.6, p = 0.011). The rate of grade 3 or 4 toxicity was 46.0%. All grade adverse events included fever, hypotension, dyspnea, confusion, hematologic changes, nausea/vomiting, fatigue, and diarrhea. In conclusion, ACT is a promising treatment modality in gynecologic cancer. We observed a particular benefit of TIL therapy and suggest inclusion of lymphodepletion in future trials.

Engineered T-cell Receptor T Cells for Cancer Immunotherapy.

Engineering immune cells to target cancer is a rapidly advancing technology. The first commercial products, chimeric-antigen receptor (CAR) T cells, are now approved for hematologic malignancies. However, solid tumors pose a greater challenge for cellular therapy, in part because suitable cancer-specific antigens are more difficult to identify and surrounding healthy tissues are harder to avoid. In addition, impaired trafficking of immune cells to solid tumors, the harsh immune-inhibitory microenvironment, and variable antigen density and presentation help tumors evade immune cells targeting cancer-specific antigens. To overcome these obstacles, T cells are being engineered to express defined T-cell receptors (TCR). Given that TCRs target intracellular peptides expressed on tumor MHC molecules, this provides an expanded pool of potential targetable tumor-specific antigens relative to the cell-surface antigens that are targeted by CAR T cells. The affinity of TCR T cells can be tuned to allow for better tumor recognition, even with varying levels of antigen presentation on the tumor and surrounding healthy tissue. Further enhancements to TCR T cells include improved platforms that enable more robust cell expansion and persistence; coadministration of small molecules that enhance tumor recognition and immune activation; and coexpression of cytokine-producing moieties, activating coreceptors, or mediators that relieve checkpoint blockade. Early-phase clinical trials pose logistical challenges involving production, large-scale manufacturing, and more. The challenges and obstacles to successful TCR T-cell therapy, and ways to overcome these and improve anticancer activity and efficacy, are discussed herein.

Mechanisms of Resistance to KRASG12C-Targeted Therapy.

KRAS mutations are among the most common drivers of human carcinogenesis, and are associated with poor prognosis and an aggressive disease course. With the advent of KRASG12C inhibitors, the RAS protein is now targetable, with such inhibitors showing marked clinical responses across multiple tumor types. However, these responses are short-lived due to the development of resistance. Preclinical studies now suggest MAPK reactivation, stimulation of CDK4/6-dependent cell-cycle transition, and immune defects as possible mechanisms of resistance. Devising strategies to overcome such resistance mechanisms, which are a barrier to long-term clinical response, remain an active area of research. SIGNIFICANCE: Although KRAS-targeted cancer therapy is revolutionary, tumors rapidly develop resistance. Understanding the mechanisms driving this resistance and designing combination strategies to overcome it are integral to achieving long-term disease control.

A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis.

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Therapeutics Targeting Mutant KRAS.

Aberrations in rat sarcoma (RAS) viral oncogene are the most prevalent and best-known genetic alterations identified in human cancers. Indeed, RAS drives tumorigenesis as one of the downstream effectors of EGFR activation, regulating cellular switches and functions and triggering intracellular signaling cascades such as the MAPK and PI3K pathways. Of the three RAS isoforms expressed in human cells, all of which were linked to tumorigenesis more than three decades ago, KRAS is the most frequently mutated. In particular, point mutations in KRAS codon 12 are present in up to 80% of KRAS-mutant malignancies. Unfortunately, there are no approved KRAS-targeted agents, despite decades of research and development. Recently, a revolutionary strategy to use covalent allosteric inhibitors that target a shallow pocket on the KRAS surface has provided new impetus for renewed drug development efforts, specifically against KRASG12C. These inhibitors, such as AMG 510 and MRTX849, show promise in early-phase studies. Nevertheless, combination strategies that target resistance mechanisms have become vital in the war against KRAS-mutant tumors.

Process Characterization and Biophysical Analysis for a Yeast-Expressed Phlebotomus papatasi Salivary Protein (PpSP15) as a Leishmania Vaccine Candidate.

Cutaneous leishmaniasis is a neglected tropical disease caused by the parasite Leishmania and transmitted by sandflies. It has become a major health problem in many tropical and subtropical countries, especially in regions of conflict and political instability. Currently, there are only limited drug treatments and no available licensed vaccine; thus, the need for more therapeutic interventions remains urgent. Previously, a DNA vaccine encoding a 15 kDa sandfly (Phlebotomus papatasi) salivary protein (PpSP15) and recombinant nonpathogenic Leishmania tarentolae secreting PpSP15 have been shown to induce protective immunity against Leishmania major in mice, demonstrating that PpSP15 is a promising vaccine candidate. In this study, we developed a fermentation process in yeast with a yield of ~1g PpSP15/L and a scalable purification process consisting of only 2 chromatographic purification steps with high binding capacity for PpSP15, suggesting that PpSP15 can be produced economically. The biophysical/biochemical analysis of the purified PpSP15 indicated that the protein was of high purity (>97%) and conformationally stable between pH 4.4 and 9.0. More importantly, the recombinant protein had a defined structure similar to that of the related PdSP15 from Phlebotomus duboscqi, implying the suitability of the yeast expression system for producing a correctly folded PpSP15.

Establishing Preferred Product Characterization for the Evaluation of RNA Vaccine Antigens.

The preferred product characteristics (for chemistry, control, and manufacture), in addition to safety and efficacy, are quintessential requirements for any successful therapeutic. Messenger RNA vaccines constitute a relatively new alternative to traditional vaccine development platforms, and thus there is less clarity regarding the criteria needed to ensure regulatory compliance and acceptance. Generally, to identify the ideal product characteristics, a series of assays needs to be developed, qualified and ultimately validated to determine the integrity, purity, stability, and reproducibility of a vaccine target. Here, using the available literature, we provide a summary of the array of biophysical and biochemical assays currently used in the field to characterize mRNA vaccine antigen candidates. Moreover, we review various in vitro functional cell-based assays that have been employed to facilitate the early assessment of the biological activity of these molecules, including the predictive immune response triggered in the host cell. Messenger RNA vaccines can be produced rapidly and at large scale, and thus will particularly benefit from well-defined and well-characterized assays ultimately to be used for in-process, release and stability-indications, which will allow equally rapid screening of immunogenicity, efficacy, and safety without the need to conduct often lengthy and costly in vivo experiments.

A method to probe protein structure from UV absorbance spectra.

Proteins primarily absorb UV light due to the presence of tryptophan, tyrosine, and phenylalanine residues, with absorbance maxima at 280, 275, and 258 nm, respectively. We now demonstrate that a simple value obtained by relating the absorbance at all three wavelengths, [A280/A275 + A280/A258], is a generally useful, robust, and sensitive probe of protein 'foldedness', and thus can be used to investigate unfolding, refolding, disulfide bonds, stability, buffer excipients, and even protein-protein and protein-ligand interactions.

Characterization and Stability of Trypanosoma cruzi 24-C4 (Tc24-C4), a Candidate Antigen for a Therapeutic Vaccine Against Chagas Disease.

Chagas disease due to chronic infection with Trypanosoma cruzi is a neglected cause of heart disease, affecting approximately 6-10 million individuals in Latin America and elsewhere. T. cruzi Tc24, a calcium-binding protein in the flagellar pocket of the parasite, is a candidate antigen for an injectable therapeutic vaccine as an alternative or a complement to chemotherapy. Previously, we reported that a genetically engineered construct from which all cysteine residues had been eliminated (Tc24-C4) yields a recombinant protein with reduced aggregation and improved analytical purity in comparison to the wild-type form, without compromising antigenicity and immunogenicity. We now report that the established process for producing Escherichia coli-expressed Tc24-C4 protein is robust and reproducibly yields protein lots with consistent analytical characteristics, freeze-thaw, accelerated, and long-term stability profiles. The data indicate that, like most proteins, Tc24-C4 should be stable at -80°C, but also at 4°C and room temperature for at least 30 days, and up to 7-15 days at 37°C. Thus, the production process for recombinant Tc24-C4 is suitable for Current Good Manufacturing Practice production and clinical testing, based on process robustness, analytical characteristics, and stability profile.

Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.

From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.

DSF Guided Refolding As A Novel Method Of Protein Production.

The Anfinsen hypothesis, the demonstration of which led to the Nobel prize in Chemistry, posits that all information required to determine a proteins' three dimensional structure is contained within its amino acid sequence. This suggests that it should be possible, in theory, to fold any protein in vitro. In practice, however, protein production by refolding is challenging because suitable refolding conditions must be empirically determined for each protein and can be painstaking. Here we demonstrate, using a variety of proteins, that differential scanning fluorimetry (DSF) can be used to determine and optimize conditions that favor proper protein folding in a rapid and high-throughput fashion. The resulting method, which we deem DSF guided refolding (DGR), thus enables the production of aggregation-prone and disulfide-containing proteins by refolding from E. coli inclusion bodies, which would not normally be amenable to production in bacteria.

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.

Structural basis for intersubunit signaling in a protein disaggregating machine.

ClpB is a ring-forming, ATP-dependent protein disaggregase that cooperates with the cognate Hsp70 system to recover functional protein from aggregates. How ClpB harnesses the energy of ATP binding and hydrolysis to facilitate the mechanical unfolding of previously aggregated, stress-damaged proteins remains unclear. Here, we present crystal structures of the ClpB D2 domain in the nucleotide-bound and -free states, and the fitted cryoEM structure of the D2 hexamer ring, which provide a structural understanding of the ATP power stroke that drives protein translocation through the ClpB hexamer. We demonstrate that the conformation of the substrate-translocating pore loop is coupled to the nucleotide state of the cis subunit, which is transmitted to the neighboring subunit via a conserved but structurally distinct intersubunit-signaling pathway common to diverse AAA+ machines. Furthermore, we found that an engineered, disulfide cross-linked ClpB hexamer is fully functional biochemically, suggesting that ClpB deoligomerization is not required for protein disaggregation.

Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine.

Hsp104 is a double ring-forming AAA+ ATPase, which harnesses the energy of ATP binding and hydrolysis to rescue proteins from a previously aggregated state. Like other AAA+ machines, Hsp104 features conserved cis- and trans-acting elements, which are hallmarks of AAA+ members and are essential to Hsp104 function. Despite these similarities, it was recently proposed that Hsp104 is an atypical AAA+ ATPase, which markedly differs in 3D structure from other AAA+ machines. Consequently, it was proposed that arginines found in the non-conserved M-domain, but not the predicted Arg-fingers, serve the role of the critical trans-acting element in Hsp104. While the structural discrepancy has been resolved, the role of the Arg-finger residues in Hsp104 remains controversial. Here, we exploited the ability of Hsp104 variants featuring mutations in one ring to retain ATPase and chaperone activities, to elucidate the functional role of the predicted Arg-finger residues. We found that the evolutionarily conserved Arg-fingers are absolutely essential for ATP hydrolysis but are dispensable for hexamer assembly in Hsp104. On the other hand, M-domain arginines are not strictly required for ATP hydrolysis and affect the ATPase and chaperone activities in a complex manner. Our results confirm that Hsp104 is not an atypical AAA+ ATPase, and uses conserved structural elements common to diverse AAA+ machines to drive the mechanical unfolding of aggregated proteins.