Mechanisms of Resistance to Small Molecules in Acute Myeloid Leukemia.

In recent years, great progress has been made in the therapy of AML by targeting cellular processes associated with specific molecular features of the disease. Various small molecules inhibiting FLT3, IDH1/IDH2, and BCL2 have already gained approval from the respective authorities and are essential parts of personalized therapeutic regimens in modern therapy of AML. Unfortunately, primary and secondary resistance to these inhibitors is a frequent problem. Here, we comprehensively review the current state of knowledge regarding molecular processes involved in primary and secondary resistance to these agents, covering both genetic and nongenetic mechanisms. In addition, we introduce concepts and strategies for how these resistance mechanisms might be overcome.

Refining AML Treatment: The Role of Genetics in Response and Resistance Evaluation to New Agents.

The number of treatment options for acute myeloid leukemia (AML) has greatly increased since 2017. This development is paralleled by the broad implantation of genetic profiling as an integral part of clinical studies, enabling us to characterize mutation-response, mutation-non-response, or mutation-relapse patterns. The aim of this review is to provide a concise overview of the current state of knowledge with respect to newly approved AML treatment options and the association of response, relapse and resistance with genetic alterations. Specifically, we will highlight current genetic data regarding FLT3 inhibitors, IDH inhibitors, hypomethylating agents (HMA), the BCL-2 inhibitor venetoclax (VEN), the anti-CD33 antibody conjugate gemtuzumab ozogamicin (GO) and the liposomal dual drug CPX-351.

Ibrutinib- and bortezomib-extended R-CHOP induction in elderly higher-risk patients newly diagnosed with diffuse large B-cell lymphoma - first analysis of toxicity and efficacy signals.

Diffuse large-cell B-cell lymphoma (DLBCL) is the most common lymphoid malignancy. About 30-40% of the patients will not be cured by standard Rituximab (R)-CHOP-like immune-chemotherapy, and many of them experience relapse and eventually succumb to their disease. Enhancing first-line efficacy in patients at higher risk, among them many elderly, is key to improve long-term outcomes. Numerous attempts to combine R-CHOP with targeted agents failed in large randomized phase III trials. The addition of Ibrutinib enhanced survival in younger patients, but increased toxicity across all age groups, especially in the elderly. Older DLBCL patients impose particular challenges, since they often present with more advanced disease, and exhibit treatment-relevant comorbidities. ImbruVeRCHOP trial aims at identifying patients who need that benefit from rationally augmented first-line regimens without experiencing overt toxicity and detecting their molecular signatures of response. This first analysis presents encouraging feasibility, safety, and preliminary response data in elderly high-risk DLBCL patients.

Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9.

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34+ progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2mut) cells, whereas ASXL1mut as well as DNMT3Amut cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.

Single-cell analysis based dissection of clonality in myelofibrosis.

Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

A Phase I/II first-line study of R-CHOP plus B-cell receptor/NF-κB-double-targeting to molecularly assess therapy response.

The ImbruVeRCHOP trial is an investigator-initiated, multicenter, single-arm, open label Phase I/II study for patients 61-80 years of age with newly diagnosed CD20+ diffuse large B-cell lymphoma and a higher risk profile (International Prognostic Index ≥2). Patients receive standard chemotherapy (CHOP) plus immunotherapy (Rituximab), a biological agent (the proteasome inhibitor Bortezomib) and a signaling inhibitor (the Bruton's Tyrosine Kinase-targeting therapeutic Ibrutinib). Using an all-comers approach, but subjecting patients to another lymphoma biopsy acutely under first-cycle immune-chemo drug exposure, ImbruVeRCHOP seeks to identify an unbiased molecular responder signature that marks diffuse large B-cell lymphoma patients at risk and likely to benefit from this regimen as a double, proximal and distal B-cell receptor/NF-κB-co-targeting extension of the current R-CHOP standard of care. EudraCT-Number: 2015-003429-32; ClinicalTrials.gov identifier: NCT03129828.

Hematopoietic lineage distribution and evolutionary dynamics of clonal hematopoiesis.

Clonal hematopoiesis of indeterminate potential (CHIP) occurs in an age-related manner and associates with an increased risk of hematologic cancer, atherosclerotic disease, and shorter overall survival. Little is known about the cell of origin, repartition patterns of clonal mutations within the hematopoietic differentiation tree, and its dynamics under evolutionary pressure. Using targeted sequencing, CHIP was identified in 121 out of 437 elderly individuals (27.7%). Variant allele frequencies (VAFs) of 91 mutations were studied in six peripheral blood cell fractions. VAFs were significantly higher in monocytes, granulocytes, and NK-cells compared to B- or T cells. In all cases with available bone marrow material, mutations could be identified in Lin-CD34+CD38- HSCs with subsequent expansion to myeloid primed progenitors. In 22 patients with solid cancer receiving (radio-)chemotherapy, longitudinal study of 32 mutations at 121 time points identified relative VAF changes of at least 50% in 13/32 mutations. VAFs of DNMT3A, were stable in 12/13 cases (P < .001). Cancer patients with a clonal mutation other than DNMT3A required more often red blood cell transfusions and dose reductions. Our results provide novel insights into cellular distribution of clonal mutations, their dynamics under chemotherapy, and advocate for systematic analyses for CHIP in cancer patients.

Frequent NFKBIE deletions are associated with poor outcome in primary mediastinal B-cell lymphoma.

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.

Pharmacological and genomic profiling identifies NF-κB-targeted treatment strategies for mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

New insights into the biology of molecular subtypes of diffuse large B-cell lymphoma and Burkitt lymphoma.

Diffuse large B-cell lymphoma and Burkitt lymphoma are two distinct lymphoma entities recognized by the current World Health Organization classification of lymphoid neoplasms. Although diagnostic criteria to distinguish these two subtypes have been established, a subset of cases have overlapping characteristics and therefore, the distinction can be challenging. Thus, the World Health Organization classification introduced an intermediate subtype referred as "B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma". This review summarizes our current understanding of the biology of these lymphoma subtypes.

Omacetaxine mepesuccinate for the treatment of leukemia.

INTRODUCTION: Omacetaxine mepesuccinate, formerly known as homoharringtonine, is a first-in-class cephalotaxine that has experienced phases of increasing and waning interest since its first use in traditional Chinese medicine. With activity being reported in patients with chronic myeloid leukemia (CML) resistant to currently available tyrosine kinase inhibitors, renewed interest has recently been generated. AREAS COVERED: The development of omacetaxine mepesuccinate, with emphasis on synthesis and mode of administration, is addressed. An overview on current clinical results as a single agent or within combination regimens in patients with acute myeloid leukemia (AML) and CML is given. EXPERT OPINION: Omacetaxine mepesuccinate has a unique mode of action and appreciable activity in AML and CML with generally mild nonhematologic toxicity. In patients with AML, results indicate a role within combination regimens in selected, possibly elderly patient populations. In CML, patients with resistance to tyrosine kinase inhibitors, especially due to the T315I mutation, are the most intensively studied. Despite successful results in some patients, single-agent therapy with omacetaxine mepesuccinate has resulted in modest results. However, upfront combination with tyrosine kinase inhibitor represents an attractive option due their differing mechanisms of action.

The molecular biology of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common type of malignant lymphoma. In the last few years, significant progress has been achieved in the understanding of the molecular pathogenesis of this entity. Gene expression profiling has identified three molecular DLBCL subtypes, termed germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). In this review, we summarize our current understanding of the biology of these DLBCL subtypes with a special emphasis on novel diagnostic and therapeutic approaches.