A number of studies presented at the 22nd Congress of EHA evaluate new therapeutic strategies in myeloid malignancies, such as targeting autophagy. One study demonstrated a role for low MYBL2 expression in the pathogenesis of myelodysplastic syndrome (MDS), another showed that the JAK2-V617F mutation causes a ribosomal frameshifting defect. Clinical studies assessed the value of sorafenib maintenance and the IDH2 inhibitor enasidenib in acute myeloid leukemia (AML), and of reducing therapy before completely stopping in chronic myeloid leukemia (CML). Measuring FLT3-ITD ratio by next generation sequencing (NGS) may be used for minimal residual disease (MRD) detection, as was presented in Madrid. In the Plenary Sessions overviews were given on new developments in myeloproliferative neoplasms (MPN), and on the behavior of hematopoietic stem cells (HSC) in their niche.
Maintenance treatment with sorafenib provides a clinically relevant survival benefit to AML patients carrying the FLT3 internal tandem duplication (FLT3-ITD). At the EHA annual congress Dr Betül Oran (MD Anderson Cancer Center, Houston, USA) presented the results of a retrospective study on behalf of her co-authors.2
Patients with FLT3-ITD AML have shorter remission durations and higher relapse rates. Although allogeneic stem cell transplantation (ASCT) improves disease-free survival, they still relapse more often with extremely poor outcomes.
The tyrosine kinase inhibitor (TKI) sorafenib targets multiple kinases, among which FLT3-ITD, and has been studied in frontline and relapse settings in AML. The aim of this study was to compare the outcomes of AML patients carrying a FLT3-ITD who received sorafenib maintenance following ASCT to those who received no maintenance at all. In order to minimize selection bias the investigators exactly matched cases and controls (1:2), ending up with 13 cases and 26 controls.
The median time to initiation of sorafenib was 55 days. At 24 months post ASCT the PFS with sorafenib maintenance was 82%, compared to 45% in the control group (p=0,1). OS was significantly better with maintenance (100%), versus 60% in the control group (p=0,035). Only 3 out of 13 cases relapsed, compared to 11 out of 26 controls. The maintenance treatment was well tolerated.
“These results are encouraging, but prospective clinical trials should define what regimen is the best, what the most tolerable dose is and who should be the target group”, concluded Oran.
The presence of MRD in AML patients after treatment may predict for relapse. In relapsed or refractory (R/R) AML patients carrying FLT3 mutations deep molecular responses to the FLT3 inhibitor gilteritinib were associated with better survival, as was presented by Dr Jessica Altman (Robert H Lurie Comprehensive Cancer Center, Chicago, USA) at the EHA annual congress.3
Gilteritinib is a potent inhibitor of FLT3 and AXL. The phase 1/2 study CHRYSALIS evaluated the tolerability and antileukemic activity of gilteritinib in an R/R AML population enriched for FLT3-ITD.4
FLT3-mutated patients demonstrated the best overall response rate and longest median OS at doses of ≥80 mg per day. Gilteritinib was well tolerated. Diarrhea, fatigue and elevated liver enzymes were the most common treatment-related adverse events.
Altman and colleagues used next generation sequencing (NGS) to quantify the FLT3-ITD signal ratio of 80 FLT3-ITD-positive patients enrolled in the 120 and 200 mg/day dose cohorts of the CHRYSALIS study. A molecular response (ITD signal ratio ≤10-2) was achieved by 25% of patients, 23% achieved a major molecular response (ITD signal ratio ≤10-3), and 16% were MRD negative (ITD signal ratio ≤10-4). The median OS of patients who had a molecular response was significantly prolonged compared to patients who did not achieve a molecular response (59,6 vs. 28,4 months, p=0.001).
“There is now a sensitive, specific assay for the detection of MRD in FLT3-ITD-mutated AML, that has the potential to be utilized”, stated Altman. Two ongoing phase 3 studies with gilteritinib are prospectively evaluating MRD as a predictive marker for clinical benefit.
Treatment with enasidenib, a novel inhibitor of mutant (m) IDH2, was well tolerated and induced durable responses in mainly older patients with R/R AML. At the EHA annual congress Dr Eytan Stein (Memorial Sloan Kettering Cancer Center, New York, USA) presented results of a phase 1/2 study with enasidenib.5
Approximately 12% of AML patients have a mutation in IDH2, resulting in production of 2-hydroxyglutarate (2HG), altered DNA methylation, and a block in myeloid differentiation. Enasidenib selectively inhibits mIDH2 and induces differentiation of leukemic cells. In a phase 1/2 dose escalation study Stein and co-investigators evaluated the tolerability, safety and clinical activity of enasidenib in patients with advanced hematologic malignancies and mIDH2, who relapsed or were refractory to prior therapy. In Madrid he reported updated results on 281 patients with R/R AML of whom 214 received 100 mg/day of enasidenib.
Median age was 68 years, most patients were considered as high risk. The treatment was well tolerated. The most common grade 3-4 treatment-related adverse event was blood bilirubin increase (8%), which according to Stein was not due to liver damage but to an off-target effect. IDH differentiation syndrome occurred in 7% of patients.
The overall response rate (ORR) was 38%, with 20% of patients achieving complete remission (CR). The median time to first response was 1.9 months, median time to CR was 3.8 months. Stein proposed that people should receive at least four cycles of therapy before deciding whether it is working or not. With a median follow-up of 6.6 months the median OS was 8.4 months. Patients who achieved CR had a median OS of 22.9 months, responders who did not reach CR still had a median OS of 15.1 months.
Ongoing studies are comparing enasidenib monotherapy to conventional care regimens and evaluating enasidenib combination therapy in newly diagnosed AML.
Multiple studies have evaluated whether patients with CML in deep molecular response (MR4; BCR-ABL1/ABL1 ratio ≤0.01%) can safely discontinue treatment with imatinib or second generation tyrosine kinase inhibitors (TKIs). The DESTINY study now shows that dose reduction prior to complete stopping results in a better relapse-free survival (RFS), as was presented by Dr Richard Clark (University of Liverpool, UK) at the Congress in Madrid.
In addition to 125 CML patients in MR4 the DESTINY study included 49 patients showing a stable, major molecular response (MMR; <0.1 but >0.01%). The patients first received TKI treatment at half dose for one year, with monthly PCR-monitoring, and when molecular response remained stable they discontinued treatment altogether for two years, with bi-monthly PCR-monitoring. At the time of the analysis, all patients who remained on study had reached the 2-year time point, and about 30 patients had completed the study.
At 24 months, including 12 months of stopping, patients in stable MR4 had a RFS of 76%. This appears to be better than the results after one year of stopping in comparable studies, such as EUROSKI, and Clark suggested that this is due to the 12 months of dose reduction prior to stopping.
RFS was 39% for patients in stable MMR. “This is less, but still not zero”, said Clark. He thinks that with a bit of caution dose reduction and stopping could be considered for these patients. Most patients who restarted treatment upon relapse achieved MMR within four months. Both dose reduction and stopping improved adverse events.
Leukemic primitive populations have higher autophagy levels than more differentiated cells. Inhibition of autophagy with the lysosomotropic agent Lys05, in combination with TKIs, may be a new therapeutic strategy for eliminating CML stem cells, as was demonstrated by Dr Pablo Baquero (University of Glasgow, UK) in his presentation at the EHA annual congress.7
Autophagy is a catabolic process that cells use to recycle cytoplasmic material. CML stem cells are insensitive to TKIs, and persistent leukemic stem cells (LSCs) are the main cause of relapse of the disease. The autophagy inhibitor hydroxychloroquine (HCQ) sensitizes LSCs to TKIs, but is not potent enough in clinical studies.
Baquero and colleagues developed second generation lysosomotropic agents. In CML progenitor cells, Lys05 was a more potent inhibitor of autophagy than HCQ, and induced differentiation of LSCs. In a mouse model of CML, the investigators observed that autophagy levels were high in primitive cell populations. Treatment of these mice with Lys05 resulted in lower levels of autophagy, reduced numbers of the more primitive LSCs, and increased numbers of LSK progenitor cells. This shows that Lys05 induces differentiation of LSCs to a more progenitor phenotype in vivo.
In the PDX mouse model human CD34+ CML cells were transplanted into irradiated, immunodeficient NSG mice. Treatment with a combination of Lys05 and the TKI nilotinib effectively eliminated LSCs, enhancing the effect of nilotinib alone. The tolerability appeared to be good as the mice did not lose any weight. “This provides a rationale to take this compound into the clinic”, concluded Baquero.
Mice with reduced levels of MYBL2 develop MDS-like disease. MDS-patients with reduced expression of MYBL2 in CD34+ bone marrow cells have a worse prognosis. At the EHA congress Dr Paloma Garcia (University of Birmingham, UK) and colleagues showed a novel role for low expression of MYBL2 in the pathogenesis of MDS by affecting the double strand break (DSB) DNA repair pathways.8
MYBL2 is a transcription factor involved in DNA replication fork progression and maintenance of genome integrity. Garcia and colleagues generated a mouse model, Mybl2+/Δ, expressing approximately half of the normal levels of MYBL2. They assessed the response of HSCs from young (7 weeks) and old (70 weeks) mice to 2 Gy ionizing radiation.
HSCs from Mybl2+/Δ mice displayed limited proliferative potential and increased sensitivity to ionizing radiation as compared to HSCs from wild-type mice. This effect increased with age. The investigators also saw longer retention of 53BP1 foci, and less Rad51 foci, which form at sites of DNA damage. 53BP1 is involved in the error-prone non-homologous-end-joining (NHEJ) and Rad51 in the error-free homologous recombination (HR) DNA repair pathway.
After ionizing irradiation the blood cell progeny of Mybl2+/Δ mice had a higher percentage of chromatids with fragile telomeres compared to wild-type mice, indicative of increased genomic instability. In addition, in MDS patients low MYBL2 expression was associated with low levels of DNA repair genes. The investigators suggest that low MYBL2 expression could contribute to the emergence of further genetic abnormalities by deregulation of DNA repair pathways.
The JAK2-V617F mutation results in increased activity of JAK2, and is a driver mutation in several MPN. The mutation not only results in increased phosphorylation of JAK2, but also in increased expression through a ribosomal frameshifting defect, as was presented by Dr Sergey Sulima (KU Leuven, Belgium).9
Increased autophosphorylation of JAK2 only partially explains the strong activation of the JAK-STAT pathway caused by JAK2-V617F. Programmed -1 ribosomal frameshifting (-1 PRF) has recently been discovered to regulate the expression of many genes, including cytokine receptors. This process lets ribosomes slip by one base, resulting in a new reading frame with premature termination codons and destabilization of the mRNA, providing an extra level of control by limiting gene expression.
The investigators found that the JAK2-V617F mutation lies within a -1 PRF signal, and disrupts ribosomal frameshifting. This results in less destabilization of JAK2 mRNA, and increased protein translation. The introduction of silent mutations in the -1 PRF signal that reduced frameshifting mimicked the increased JAK2 expression and transforming activity of V617F, albeit to a lesser extent. Injection of cells carrying these silent mutations or V617F into mice resulted in a similar leukemia phenotype, although leukemia occurred faster with V617F.
Three additional -1 PRF signals are present in the JAK2 mRNA, and JAK2-V617F homozygous patients have increased levels of JAK2 mRNA, further supporting a role for ribosomal frameshifting in controlling JAK2 protein production. Targeting the kinase activity as well as the expression of JAK2 may provide a new treatment strategy for MPN.
In the Plenary Session on Sunday Dr Radek Skoda (University Hospital Basel, Switzerland) gave an overview of recent, unpublished discoveries in his lab and how they might contribute to the pathogenesis of MPN.
Using genetic linkage analysis and sequencing Skoda and co-workers identified a novel mutation of the erythropoietin (EPO) gene in a family with erythrocytosis, where a mutation in exon 2 results in a frameshift. Although the disease phenotype predicts a gain of function mutation, expression of the mutated EPO cDNA in a cell line resulted in a loss of function, with markedly reduced levels of the mutated protein. The investigators then demonstrated that an alternative transcript of EPO exists that normally does not produce any protein. However, with the single nucleotide deletion this non-coding transcript becomes in frame with the coding sequence and produces EPO. This transcript is responsible for overproduction of EPO and erythrocytosis.
Another discovery came from the observation that mice expressing mutant Jak2 have reduced body weight and almost no fat tissue, although they ate more and exercised less than wildtype mice. The mice had reduced white adipose tissue, and showed severe hypoglycemia. Treatment with ruxolitinib abrogated the changes in metabolism, resulting in increased fasting glucose levels. A high-fat diet hardly increased the body weight of the mutant mice, but significantly improved survival. Inhibition of fatty acid oxidation led to decreased survival of MPN mice, suggesting dependence on fatty acids and lypolysis, which may provide new targets for therapy.
In the Plenary Session on Saturday Dr David Scadden (Harvard University, Cambridge, MA, USA) discussed the fundamental ways in which blood cell production is regulated. “This is of relevance not just for normal cells, but may also help us understand how we respond to the setting in which genetic alterations occur”, he said.
According to one hypothesis the stem cell niche provides an environment to stem cells that enables their sustained unlimited proliferation and regulates the maturation constraint. Multiple cell types have been shown play a role in this niche, such as neural, mesenchymal, endothelial cells, and descendant cells of the HSC. However, another hypothesis proposes that the regulation of HSCs is cell autonomous.
Scadden's group developed a mouse model, the ‘Hue’ mouse, that enables clonal tracking of HSCs and their descendant cells with different fluorescent colors. In bone marrow stroma they observed fields of cells with the same color, suggesting that the stroma does provide environmental cues, and supporting the notion that genetic mutations in stroma can provide an abnormal field.
To determine whether HSCs have their own intrinsic behavior, or whether the microenvironment dictates their fate, they transplanted HSCs from Hue mice into different recipient mice. The HSCs had clone specific behaviors that were similar in all recipient mice, and were preserved under conditions of stress. The clonal behavior was associated with the epigenetic, but not transcriptional state of the HSC. The investigators conclude that HSCs are epigenetically constrained with limited functional plasticity in response to environmental stress.