Patients with TP53-Mutated Acute Myeloid Leukemia Receiving Intensive Induction Therapy Have Superior Outcomes Due to a Higher Rate of Salvage Therapy: A Single Institution, Retrospective Study.

BACKGROUND: TP53 mutations (TP53m) define the most treatment-refractory acute myeloid leukemia (AML) subtype. Optimal treatment approaches have not been established in this setting. We reviewed our institutional experience to identify therapy sequencing, treatment response, and survival patterns in these patients. METHODS: This study was a single-center, retrospective cohort analysis. RESULTS: Our cohort includes 86 TP53m and 337 TP53 wild-type (TP53wt) adult AML patients. TP53m AML patients presented with lower bone marrow and peripheral blasts; none presented with hyperleukocytosis. Patients who received intensive treatment up front demonstrated superior overall survival (OS) over those receiving first-line non-intensive therapy (2-year OS 22% versus 7%; p = 0.02). However, the complete remission (CR) rates among the first-line intensive and non-intensive therapy groups were comparable (21.9% and 29.4%, respectively, p = 0.49). The improved OS is therefore attributed to superior cumulative CR in the intensive group. First-line intensively treated patients were more likely to receive and respond to salvage, leading to a cumulative CR rate of 65.7% (versus 29.4%, p = 0.003). Achieving CR at any point is strongly associated with superior survival outcomes with 2-year OS of 31% versus 0% for those not achieving CR ever (p < 0.01). CONCLUSIONS: We find that TP53m AML rarely presents with oncological emergencies, suggesting that clinical trial enrollment is feasible in this group. Additionally, in our cohort, intensive induction therapies lead to superior survival outcomes attributed to successful salvage therapy. These data suggest that strategic therapy sequencing and salvage therapy may be important in optimizing outcomes for TP53m AML patients.

R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer. SIGNIFICANCE: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma.

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

CD69 marks a subpopulation of acute myeloid leukemia with enhanced colony forming capacity and a unique signaling activation state.

In acute myeloid leukemia (AML), leukemia stem cells (LSCs) have self-renewal potential and are responsible for relapse. We previously showed that, in Mll-AF9/NRASG12V murine AML, CD69 expression marks an LSC-enriched subpopulation with enhanced in vivo self-renewal capacity. Here, we used CyTOF to define activated signaling pathways in LSC subpopulations in Mll-AF9/NRASG12V AML. Furthermore, we compared the signaling activation states of CD69High and CD36High subsets of primary human AML. The human CD69High subset expresses low levels of Ki67 and high levels of NFκB and pMAPKAPKII. Additionally, the human CD69High AML subset also has enhanced colony-forming capacity. We applied Bayesian network modeling to compare the global signaling network within the human AML subsets. We find that distinct signaling states, distinguished by NFκB and pMAPKAPKII levels, correlate with divergent functional subsets, defined by CD69 and CD36 expression, in human AML. Targeting NFκB with proteasome inhibition diminished colony formation.

Immunophenotypically-defined murine AML stem cells harbor self-renewing and non-self-renewing subsets that display unique signaling characteristics.CD69, an NFκB target gene, marks a subset of human AML with increased colony forming capacity and reduced proliferation.NFκB activation correlates with the global signaling pathway activation state in human AML.

Lymphomatous infiltration of the kidney in a patient with Waldenstrom's macroglobulinemia.

Kidney disease can be an initial presentation or a chronic manifestation of plasma cell dyscrasias. Here, we describe a rare presentation of kidney disease driven by lymphomatous infiltration of the kidney in a patient with Waldenstrom's macroglobulinemia (WM). A 70-year-old female with an 8-year history of WM (IgM, κ) was referred for declining renal function. Prior to presentation, she had stable WM disease without evidence of worsening disease burden. She had been previously hospitalized with SARS-CoV-2 infection and acute kidney injury (AKI). Her serum creatinine (sCr) peaked at 3.7 mg/dL (baseline 0.9 mg/dL) but recovered to 1.1 mg/dL by the time of discharge. Two months after discharge, her sCr increased to 1.9 mg/dL, and she had new proteinuria of 1.5 g/day. Kidney biopsy showed lymphomatous infiltration of the interstitium without glomerular involvement. Treatment with rituximab and bendamustine resulted in an improvement in renal function (sCr 1.4 mg/dL). WM is an uncommon hematologic malignancy, and extramedullary involvement, including renal involvement, is rare. This case emphasizes the importance of surveillance for kidney dysfunction in patients with plasma cell dyscrasias, even if patients appear to have stable lymphoproliferative disease.

Identification of NRAS Downstream Genes with CRISPR Activation Screening.

Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

Proliferation and Self-Renewal Are Differentially Sensitive to NRASG12V Oncogene Levels in an Acute Myeloid Leukemia Cell Line.

NRAS proteins are central regulators of proliferation, survival, and self-renewal in leukemia. Previous work demonstrated that the effects of oncogenic NRAS in mediating proliferation and self-renewal are mutually exclusive within leukemia subpopulations and that levels of oncogenic NRAS vary between highly proliferative and self-renewing leukemia subpopulations. These findings suggest that NRAS activity levels may be important determinants of leukemic behavior. To define how oncogenic NRAS levels affect these functions, we genetically engineered an acute myeloid leukemia (AML) cell line, THP-1, to express variable levels of NRASG12V. We replaced the endogenous NRASG12D gene with a tetracycline-inducible and dose-responsive NRASG12V transgene. Cells lacking NRASG12V oncoprotein were cell-cycle arrested. Intermediate levels of NRASG12V induced maximal proliferation; higher levels led to attenuated proliferation, increased G1 arrest, senescence markers, and maximal self-renewal capacity. Higher levels of the oncoprotein also induced self-renewal and mitochondrial genes. We used mass cytometry (CyTOF) to define the downstream signaling events that mediate these differential effects. Not surprisingly, we found that the levels of such canonical RAS-effectors as pERK and p4EBP1 correlated with NRASG12V levels. ß-Catenin, a mediator of self-renewal, also correlated with NRASG12V levels. These signaling intermediates may mediate the differential effects of NRASG12V in leukemia biology. Together, these data reveal that oncogenic NRAS levels are important determinants of leukemic behavior explaining heterogeneity in phenotypes within a clone. This system provides a new model to study RAS oncogene addiction and RAS-induced self-renewal in AML. IMPLICATIONS: Different levels of activated NRAS may exert distinct effects on proliferation and self-renewal.

High risk of relapse with intermediate dose cytarabine for consolidation in young favourable-risk acute myeloid leukaemia patients following induction with 7+3: a retrospective multicentre analysis and critical review of the literature.

Following the 2017 European LeukemiaNet (ELN) guidelines, we changed our practice from using high-dose cytarabine (HIDAC-3 g/m2 q12h-D1,3,5) to intermediate-dose cytarabine (IDAC-1·5 g/m2 q12h-D1,3,5/D1-3) for consolidation in young(<60 years) favourable-risk acute myeloid leukaemia (AML) patients. We assessed the clinical impact of this practice change. Of 80 patients, 51 received HIDAC prior to the protocol change, and subsequently, 29 received IDAC. The three-year risk of relapse was significantly higher with IDAC [61%; 95% confidence interval (CI) 40-82] compared with HIDAC (22%; 10-34), P < 0·01. Our findings suggest HIDAC, rather than IDAC, is the preferred dose for single-agent cytarabine consolidation in young, favourable-risk AML following 7+3 induction.

Primary Cardiac Lymphoma: Three Case Reports and a Review of the Literature.

Primary cardiac lymphoma (PCL) is a rare entity that comprises only 1-2% of all cardiac tumors. Due to their scarcity and variable clinical presentation, early diagnosis is challenging. In this series, three cases of PCL from a single institution are described, which highlight the spectrum of presenting features and emphasize common principles. In the first case, a 73-year-old male who presented with dyspnea was found to have a 12.1 cm mass in the right ventricle. Biopsy via cardiac catheterization revealed diffuse large B cell lymphoma (DLBCL). He was treated with chemoimmunotherapy and survived for two months. The second case describes a 55-year-old female who presented with chest pain. Imaging revealed a 3.1 cm right atrial mass and bilateral pleural effusions, with cytology from the latter demonstrating DLBCL. She was lost to follow up after three cycles of chemoimmunotherapy. In the last case, an 80-year-old female presented with weakness. A 4.0 cm mass was discovered in the right atrium and the patient expired shortly after admission. Autopsy confirmed the diagnosis of DLBCL. These case summaries are followed by a review of the clinical presentation, diagnostic approach, and treatment outcomes of PCL.

Prognostic factors for clinical outcomes of patients with central nervous system leukemia.

Prognostic factors associated with clinical outcomes of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients with central nervous system (CNS) involvement are unknown. We retrospectively studied the characteristics and outcomes of 66 (18 pediatric and 48 adult) patients with CNS leukemia with ALL (n = 41) or AML (n = 25). The median age of patients at diagnosis of CNS leukemia was 30 (range, 1-69) years. Nearly two-third patients had CNS involvement at the initial diagnosis of leukemia. Complete remission of CNS leukemia was attained in 58 (88%) patients, and probability of overall survival at 36 months after the diagnosis of CNS leukemia was 43% for the entire cohort. We identified that achieving remission of systemic leukemia and having CNS leukemia diagnosed and treated before allogeneic transplantation were the factors associated with CNS leukemia remission. Prognostic factors associated with better overall survival in patients with CNS leukemia included pediatric age, diagnosis of CNS leukemia before receiving allogenic transplantation, achieving clearance of systemic or CNS leukemia, receiving no cranial radiation in conjunction with intrathecal chemotherapy (IT), and receiving IT consolidation after achieving remission of CNS leukemia. Our findings show that patients with CNS leukemia are at considerable risk of mortality. Awareness of modifiable prognostic factors such as avoidance of cranial radiation whenever possible and use of IT consolidation can result in improved outcomes in subset of patients with CNS leukemia.

Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia.

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Multiomic Profiling of Tyrosine Kinase Inhibitor-Resistant K562 Cells Suggests Metabolic Reprogramming To Promote Cell Survival.

Resistance to chemotherapy can occur through a wide variety of mechanisms. Resistance to tyrosine kinase inhibitors (TKIs) often arises from kinase mutations-however, "off-target" resistance occurs but is poorly understood. Previously, we established cell line resistance models for three TKIs used in chronic myeloid leukemia treatment, and found that resistance was not attributed entirely to failure of kinase inhibition. Here, we performed global, integrated proteomic and transcriptomic profiling of these cell lines to describe mechanisms of resistance at the protein and gene expression level. We used whole transcriptome sequencing and SWATH-based data-independent acquisition mass spectrometry (DIA-MS), which does not require isotopic labels and provides quantitative measurements of proteins in a comprehensive, unbiased fashion. The proteomic and transcriptional data were correlated to generate an integrated understanding of the gene expression and protein alterations associated with TKI resistance. We defined mechanisms of resistance and two novel markers, CA1 and alpha-synuclein, that were common to all TKIs tested. Resistance to all of the TKIs was associated with oxidative stress responses, hypoxia signatures, and apparent metabolic reprogramming of the cells. Metabolite profiling and glucose-dependence experiments showed that resistant cells had routed their metabolism through glycolysis (particularly through the pentose phosphate pathway) and exhibited disruptions in mitochondrial metabolism. These experiments are the first to report a global, integrated proteomic, transcriptomic, and metabolic analysis of TKI resistance. These data suggest that although the mechanisms are complex, targeting metabolic pathways along with TKI treatment may overcome pan-TKI resistance.

Novel single-cell technologies in acute myeloid leukemia research.

Acute myeloid leukemia (AML) is a lethal malignancy because patients who initially respond to chemotherapy eventually relapse with treatment refractory disease. Relapse is caused by leukemia stem cells (LSCs) that reestablish the disease through self-renewal. Self-renewal is the ability of a stem cell to produce copies of itself and give rise to progeny cells. Therefore, therapeutic strategies eradicating LSCs are essential to prevent relapse and achieve long-term remission in AML. AML is a heterogeneous disease both at phenotypic and genotypic levels, and this heterogeneity extends to LSCs. Classical studies in AML have aimed at characterization of the bulk tumor population, thereby masking cellular heterogeneity. Single-cell approaches provide a novel opportunity to elucidate molecular mechanisms in heterogeneous diseases such as AML. In recent years, major advancements in single-cell measurement systems have revolutionized our understanding of the pathophysiology of AML and enabled the characterization of LSCs. Identifying the molecular mechanisms critical to AML LSCs will aid in the development of targeted therapeutic strategies to combat this deadly disease.

Human Melanoma-Derived Extracellular Vesicles Regulate Dendritic Cell Maturation.

Evolution of melanoma from a primary tumor to widespread metastasis is crucially dependent on lymphatic spread. The mechanisms regulating the initial step in metastatic dissemination via regional lymph nodes remain largely unknown; however, evidence supporting the establishment of a pre-metastatic niche is evolving. We have previously described a dysfunctional immune profile including reduced expression of dendritic cell (DC) maturation markers in the first node draining from the primary tumor, the sentinel lymph node (SLN). Importantly, this phenotype is present prior to evidence of nodal metastasis. Herein, we evaluate melanoma-derived extracellular vesicles (EVs) as potential mediators of the premetastatic niche through cargo-specific polarization of DCs. DCs matured in vitro in the presence of melanoma EVs demonstrated significantly impaired expression of CD83 and CD86 as well as decreased expression of Th1 polarizing chemokines Flt3L and IL15 and migration chemokines MIP-1α and MIP-1ß compared to liposome-treated DCs. Profiling of melanoma EV cargo identified shared proteomic and RNA signatures including S100A8 and S100A9 protein cargo, which in vitro compromised DC maturation similar to melanoma EVs. Early evidence demonstrates that similar EVs can be isolated from human afferent lymphatic fluid ex vivo. Taken together, here, we propose melanoma EV cargo as a mechanism by which DC maturation is compromised warranting further study to consider this as a potential mechanism enabled by the primary tumor to establish the premetastatic niche in tumor-draining SLNs of patients.

Phenotypic and functional characterization of a bortezomib-resistant multiple myeloma cell line by flow and mass cytometry.

Multiple myeloma (MM) is an incurable malignant plasma cell neoplasm. Proteasome inhibitors including Bortezomib (Bz) are used to treat MM, and treatment failure due to drug resistance occurs. Bz-sensitive and -resistant MM cells have distinct immunophenotypic signatures that correlate with clinical outcome. These changes can be identified by fluorescence-based cytometry (FBC), however, FBC is rarely used in predicting Bz resistance. Mass cytometry (MC) is a recently developed variation of flow cytometry that detects heavy metal-ion tagged antibodies using time-of-flight mass spectrometry allowing for detection of up to 38 epitopes simultaneously in a single cell, without significant overlap, exceeding the dimensionality of FBC 3-4-fold. Here, we compared FBC and MC in the immunophenotypic characterization of Bz-sensitive and -resistant human MM cell line U266. We show that Bz-resistant cells are associated with the loss of CD56 and CD66a adhesion molecules as well as an activation signature.