Mass Spectrometry-Based Proteogenomics: New Therapeutic Opportunities for Precision Medicine.

Proteogenomics refers to the integration of comprehensive genomic, transcriptomic, and proteomic measurements from the same samples with the goal of fully understanding the regulatory processes converting genotypes to phenotypes, often with an emphasis on gaining a deeper understanding of disease processes. Although specific genetic mutations have long been known to drive the development of multiple cancers, gene mutations alone do not always predict prognosis or response to targeted therapy. The benefit of proteogenomics research is that information obtained from proteins and their corresponding pathways provides insight into therapeutic targets that can complement genomic information by providing an additional dimension regarding the underlying mechanisms and pathophysiology of tumors. This review describes the novel insights into tumor biology and drug resistance derived from proteogenomic analysis while highlighting the clinical potential of proteogenomic observations and advances in technique and analysis tools.

Phase Ib study of sabatolimab (MBG453), a novel immunotherapy targeting TIM-3 antibody, in combination with decitabine or azacitidine in high- or very high-risk myelodysplastic syndromes.

The safety and efficacy of sabatolimab, a novel immunotherapy targeting T-cell immunoglobulin domain and mucin domain-3 (TIM-3), was assessed in combination with hypomethylating agents (HMAs) in patients with HMA-naive revised International Prognostic System Score (IPSS-R) high- or very high-risk myelodysplastic syndromes (HR/vHR-MDS) or chronic myelomonocytic leukemia (CMML). Sabatolimab + HMA had a safety profile similar to that reported for HMA alone and demonstrated durable clinical responses in patients with HR/vHR-MDS. These results support the ongoing evaluation of sabatolimab-based combination therapy in MDS, CMML, and acute myeloid leukemia.

Functional genomic landscape of acute myeloid leukaemia.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.

Cytogenetically Cryptic Acute Promyelocytic Leukemia: A Diagnostic Challenge.

Cytogenetically cryptic acute promyelocytic leukemia (APL) is rare, characterized by typical clinical and morphological features, but lacks t(15;17)(q24;q21)/PML::RARA translocation seen in conventional karyotyping or FISH. The prompt diagnosis and treatment of APL are critical due to life-threatening complications associated with this disease. However, cryptic APL cases remain a diagnostic challenge that could mislead the appropriate treatment. We describe four cryptic APL cases and review reported cases in the literature. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most efficient diagnostic modality to detect these cases, and alternative methods are also discussed. This study highlights the importance of using parallel testing methods to diagnose cryptic APL cases accurately and effectively.

Proteomic and phosphoproteomic measurements enhance ability to predict ex vivo drug response in AML.

Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemic cells with diverse mutation spectra, which eventually leads to disease relapse. This genetic heterogeneity drives the activation of complex signaling pathways that is reflected at the protein level. This diversity makes it difficult to treat AML with targeted therapy, requiring custom patient treatment protocols tailored to each individual's leukemia. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify genomic signatures that could predict patient-specific drug responses. However, there are inherent weaknesses in using only genetic and transcriptomic measurements as surrogates of drug response, particularly the absence of direct information about phosphorylation-mediated signal transduction. As a member of the Clinical Proteomic Tumor Analysis Consortium, we have extended the molecular characterization of this cohort by collecting proteomic and phosphoproteomic measurements from a subset of these patient samples (38 in total) to evaluate the hypothesis that proteomic signatures can improve the ability to predict response to 26 drugs in AML ex vivo samples. In this work we describe our systematic, multi-omic approach to evaluate proteomic signatures of drug response and compare protein levels to other markers of drug response such as mutational patterns. We explore the nuances of this approach using two drugs that target key pathways activated in AML: quizartinib (FLT3) and trametinib (Ras/MEK), and show how patient-derived signatures can be interpreted biologically and validated in cell lines. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to assess drug sensitivity in AML.

Discovery and characterization of targetable NTRK point mutations in hematologic neoplasms.

Much of what is known about the neurotrophic receptor tyrosine kinase (NTRK) genes in cancer was revealed through identification and characterization of activating Trk fusions across many tumor types. A resurgence of interest in these receptors has emerged owing to the realization that they are promising therapeutic targets. The remarkable efficacy of pan-Trk inhibitors larotrectinib and entrectinib in clinical trials led to their accelerated, tissue-agnostic US Food and Drug Administration (FDA) approval for adult and pediatric patients with Trk-driven solid tumors. Despite our enhanced understanding of Trk biology in solid tumors, the importance of Trk signaling in hematological malignancies is underexplored and warrants further investigation. Herein, we describe mutations in NTRK2 and NTRK3 identified via deep sequencing of 185 patients with hematological malignancies. Ten patients contained a point mutation in NTRK2 or NTRK3; among these, we identified 9 unique point mutations. Of these 9 mutations, 4 were oncogenic (NTRK2A203T, NTRK2R458G, NTRK3E176D, and NTRK3L449F), determined via cytokine-independent cellular assays. Our data demonstrate that these mutations have transformative potential to promote downstream survival signaling and leukemogenesis. Specifically, the 3 mutations located within extracellular (ie, NTRK2A203T and NTRK3E176D) and transmembrane (ie, NTRK3L449F) domains increased receptor dimerization and cell-surface abundance. The fourth mutation, NTRK2R458G, residing in the juxtamembrane domain, activates TrkB via noncanonical mechanisms that may involve altered interactions between the mutant receptor and lipids in the surrounding environment. Importantly, these 4 activating mutations can be clinically targeted using entrectinib. Our findings contribute to ongoing efforts to define the mutational landscape driving hematological malignancies and underscore the utility of FDA-approved Trk inhibitors for patients with aggressive Trk-driven leukemias.

Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML.

BACKGROUND: Mutations in the gene encoding isocitrate dehydrogenase 1 ( IDH1) occur in 6 to 10% of patients with acute myeloid leukemia (AML). Ivosidenib (AG-120) is an oral, targeted, small-molecule inhibitor of mutant IDH1. METHODS: We conducted a phase 1 dose-escalation and dose-expansion study of ivosidenib monotherapy in IDH1-mutated AML. Safety and efficacy were assessed in all treated patients. The primary efficacy population included patients with relapsed or refractory AML receiving 500 mg of ivosidenib daily with at least 6 months of follow-up. RESULTS: Overall, 258 patients received ivosidenib and had safety outcomes assessed. Among patients with relapsed or refractory AML (179 patients), treatment-related adverse events of grade 3 or higher that occurred in at least 3 patients were prolongation of the QT interval (in 7.8% of the patients), the IDH differentiation syndrome (in 3.9%), anemia (in 2.2%), thrombocytopenia or a decrease in the platelet count (in 3.4%), and leukocytosis (in 1.7%). In the primary efficacy population (125 patients), the rate of complete remission or complete remission with partial hematologic recovery was 30.4% (95% confidence interval [CI], 22.5 to 39.3), the rate of complete remission was 21.6% (95% CI, 14.7 to 29.8), and the overall response rate was 41.6% (95% CI, 32.9 to 50.8). The median durations of these responses were 8.2 months (95% CI, 5.5 to 12.0), 9.3 months (95% CI, 5.6 to 18.3), and 6.5 months (95% CI, 4.6 to 9.3), respectively. Transfusion independence was attained in 29 of 84 patients (35%), and patients who had a response had fewer infections and febrile neutropenia episodes than those who did not have a response. Among 34 patients who had a complete remission or complete remission with partial hematologic recovery, 7 (21%) had no residual detectable IDH1 mutations on digital polymerase-chain-reaction assay. No preexisting co-occurring single gene mutation predicted clinical response or resistance to treatment. CONCLUSIONS: In patients with advanced IDH1-mutated relapsed or refractory AML, ivosidenib at a dose of 500 mg daily was associated with a low frequency of grade 3 or higher treatment-related adverse events and with transfusion independence, durable remissions, and molecular remissions in some patients with complete remission. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02074839 .).

Molecularly targeted drug combinations demonstrate selective effectiveness for myeloid- and lymphoid-derived hematologic malignancies.

Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.

The combination of NPM1, DNMT3A, and IDH1/2 mutations leads to inferior overall survival in AML.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease with a clinical course predicted by recurrent cytogenetic abnormalities and/or gene mutations. The NPM1 insertion mutations define the largest distinct genetic subset, ∼30% of AML, and is considered a favorable risk marker if there is no (or low allelic ratio) FLT3 internal tandem duplication (FLT3 ITD) mutation. However, ∼40% of patients with mutated NPM1 without FLT3 ITD still relapse, and the factors that drive relapse are still not fully understood. We used a next-generation sequencing panel to examine mutations at diagnosis; clearance of mutations after therapy, and gain/loss of mutations at relapse to prioritize mutations that contribute to relapse. Triple mutation of NPM1, DNMT3A and IDH1/2 showed a trend towards inferior overall survival in our discovery dataset, and was significantly associated with reduced OS in a large independent validation cohort. Analysis of relative variant allele frequencies suggests that early mutation and expansion of DNMT3A and IDH1/2 prior to acquisition of NPM1 mutation leads to increased risk of relapse. This subset of patients may benefit from allogeneic stem cell transplant or clinical trials with IDH inhibitors.

Ponatinib overcomes FGF2-mediated resistance in CML patients without kinase domain mutations.

Development of resistance to kinase inhibitors remains a clinical challenge. Kinase domain mutations are a common mechanism of resistance in chronic myeloid leukemia (CML), yet the mechanism of resistance in the absence of mutations remains unclear. We tested proteins from the bone marrow microenvironment and found that FGF2 promotes resistance to imatinib in vitro. Fibroblast growth factor 2 (FGF2) was uniquely capable of promoting growth in both short- and long-term assays through the FGF receptor 3/RAS/c-RAF/mitogen-activated protein kinase pathway. Resistance could be overcome with ponatinib, a multikinase inhibitor that targets BCR-ABL and FGF receptor. Clinically, we identified CML patients without kinase domain mutations who were resistant to multiple ABL kinase inhibitors and responded to ponatinib treatment. In comparison to CML patients with kinase domain mutations, these patients had increased FGF2 in their bone marrow when analyzed by immunohistochemistry. Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGF receptor inhibition. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance.

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism.

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70-80% of patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C-terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium-binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL-3-independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2-V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll-like receptor agonist. These effects are not dependent on the novel C-terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis.

Mapping the proteogenomic landscape enables prediction of drug response in acute myeloid leukemia.

Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently predict therapeutic response. Here, we combine transcriptomic, proteomic, and phosphoproteomic datasets with ex vivo drug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measure the proteome and phosphoproteome of 210 patients and combine them with genomic and transcriptomic measurements to identify four proteogenomic subtypes that complement existing genetic subtypes. We build a predictor to classify samples into subtypes and map them to a "landscape" that identifies specific drug response patterns. We then build a drug response prediction model to identify drugs that target distinct subtypes and validate our findings on cell lines representing various stages of quizartinib resistance. Our results show how multiomics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.

Building on Foundations: Venetoclax-Based Combinations in the Treatment of Acute Myeloid Leukemia.

Frontline acute myeloid leukemia (AML) treatment is determined by a combination of patient and genetic factors. This includes patient fitness (i.e., comorbidities that increase the risk of treatment-related mortality) and genetic characteristics, including cytogenetic events and gene mutations. In older unfit patients, the standard of care treatment is typically venetoclax (VEN) combined with hypomethylating agents (HMA). Recently, several drugs have been developed targeting specific genomic subgroups of AML patients, enabling individualized therapy. This has resulted in investigations of doublet and triplet combinations incorporating VEN aimed at overcoming known resistance mechanisms and improving outcomes in older patients with AML. These combinations include isocitrate dehydrogenase-1/2 (IDH1/2) inhibitors (i.e., ivosidenib and enasidenib), fms-like tyrosine kinase 3 (FLT3) inhibitors (i.e., gilteritinib), anti-CD47 antibodies (i.e., magrolimab), mouse double minute-2 (MDM2) inhibitors, and p53 reactivators (i.e., eprenetapopt). This review summarizes ongoing trials aimed at overcoming known VEN resistance mechanisms and improving outcomes beyond that observed with HMA + VEN combinations in the treatment of AML.

Comparison and validation of the 2022 European LeukemiaNet guidelines in acute myeloid leukemia.

Risk stratification in acute myeloid leukemia (AML) remains principle in survival prognostication and treatment selection. The 2022 European LeukemiaNet (ELN) recommendations were recently published, with notable updates to risk group assignment. The complexity of risk stratification and comparative outcomes between the 2022 and 2017 ELN guidelines remains unknown. This comparative analysis evaluated outcomes between the 2017 and 2022 ELN criteria in patients enrolled within the multicenter Beat AML cohort. Five hundred thirteen patients were included. Most patients had 1 or 2 ELN risk-defining abnormalities. In patients with ≥2 ELN risk-defining mutations, 44% (n = 132) had mutations spanning multiple ELN risk categories. Compared with ELN 2017 criteria, the updated ELN 2022 guidelines changed the assigned risk group in 15% of patients, including 10%, 26%, and 6% of patients categorized as being at ELN 2017 favorable-, intermediate-, and adverse-risk, respectively. The median overall survival across ELN 2022 favorable-, intermediate-, and adverse-risk groups was not reached, 16.8, and 9.7 months, respectively. The ELN 2022 guidelines more accurately stratified survival between patients with intermediate- or adverse-risk AML treated with induction chemotherapy compared with ELN 2017 guidelines. The updated ELN 2022 guidelines better stratify survival between patients with intermediate- or adverse-risk AML treated with induction chemotherapy. The increased complexity of risk stratification with inclusion of additional cytogenetic and molecular aberrations necessitates clinical workflows simplifying risk stratification.

IDH Inhibitors in AML-Promise and Pitfalls.

PURPOSE OF REVIEW: Mutations in isocitrate dehydrogenase genes (IDH1 and IDH2) are common in acute myeloid leukemia (AML), occurring in up to 30% of AML cases. Mutations in IDH leads to abnormal epigenetic regulation in AML cells and blocks differentiation. Inhibitors of mutated IDH1 and IDH2, ivosidenib and enasidenib, respectively, were recently approved by the FDA for relapsed/refractory AML; ivosidenib is also approved for newly diagnosed AML patients not fit for standard chemotherapy. Here, we discuss the clinical development of IDH inhibitors, their unique side effects, and outline future combination approaches in AML. RECENT FINDINGS: IDH inhibitors are well-tolerated but can induce differentiation of AML cells, which leads to the on-target side effect of differentiation syndrome in up to 20% of patients. Although IDH inhibitors demonstrate efficacy as monotherapy, recent trials have shown that they have higher response rates in combination with hypomethylating agents (HMAs). Current trials of IDH inhibitors include combination with standard induction chemotherapy, as maintenance therapy, and in combination with venetoclax-based regimens. IDH inhibitors are active and have a favorable toxicity profile in AML therapy. Current clinical trials are evaluating how to best incorporate IDH inhibitors into combination therapy to optimize outcomes and duration of response for AML patients with IDH mutations.

Engrafted Donor-Derived Clonal Hematopoiesis after Allogenic Hematopoietic Cell Transplantation is Associated with Chronic Graft-versus-Host Disease Requiring Immunosuppressive Therapy, but no Adverse Impact on Overall Survival or Relapse.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated condition defined by the presence of a somatic mutation in a leukemia-associated gene in individuals who otherwise have no evidence of a hematologic malignancy. In the allogeneic hematopoietic cell transplantation (HCT) setting, clonal hematopoiesis (CH) mutations present in donor stem cells can be transferred to recipients at the time of HCT. Given that the consequences of donor-derived CH in HCT recipients are not entirely clear, we sought to investigate clinical outcomes in patients with engrafted donor-derived CH using a matched cohort analysis of both related and unrelated donors. Of 209 patients with next-generation sequencing performed before and after HCT, donor-derived CH mutations were detected in 15 (5.2%). DNMT3A was the most commonly mutated gene (9 of 15; 60%); mutations in SF3B1, CSF3R, STAT3, CBLB, TET2, and ASXL1 were also identified. Donor-derived CH was not associated with delayed neutrophil or platelet engraftment, and there was no impact on conversion to full donor chimerism. No patients with donor-derived CH experienced relapse, in contrast to 15.6% (7 of 45) in the matched control cohort without CH (P = .176). Donor-derived CH was not associated with worse overall survival; however, patients with donor-derived CH were more likely to develop chronic graft-versus-host disease (GVHD) necessitating systemic immunosuppressive therapy (IST) (P = .045) and less likely to discontinue IST (P = .03) compared with controls without donor-derived CH. We conclude that donor-derived CH does not have an adverse impact on relapse, survival, or engraftment outcomes but may potentiate a graft-versus-leukemia effect, as reflected by increased chronic GVHD necessitating IST.