ALBERT

Description: Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Description: Mouse xenografts are routinely used for in vivo studies on human acute myeloid leukemia (AML) cells. However, a significant proportion of primary AML samples (~50-60% of cases) are not able to repopulate mice, or respectively require a particularly long time (up to 9 months) to show detectable engraftment in such models. Here we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) strongly improved the engraftment of human AML cells in NOD/SCID/IL2Rgnull (NSG) mice. Sublethally irradiated gender-matched NSG mice of 6-8 weeks of age (n=40) were transplanted with primary AML cells (n=5 patients). For each sample, equal numbers of freshly thawed AML cells were injected via the tail vein at both time-points; transplanted mice were afterwards monitored for human leukemic engraftment in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry and immunohistochemistry. In all n=5 analyzed AML patient samples, transplantation at night strongly promoted leukemogenesis, with 2/5 samples showing engraftment only in the night transplant condition and 3/5 samples showing engraftment in both but significantly higher leukemic burden in night vs. day transplanted mice (Fig. 1a), although similar leukemia-specific mutations were retrieved in mice of the two conditions by next generation sequencing analysis (Fig. 1b). Limiting dilution experiments (n=2 AML samples) further confirmed these findings and showed that in fact lower numbers of AML cells were required to initiate leukemia when cells were transplanted at night versus day times. Given that the immediate process that happens after intravenous injection of AML cells into mice involves homing to BM niches, we hypothesized that the time-point of transplantation influences engraftment by modulating homing. Therefore, we performed homing assays as described for healthy hematopoietic stem/progenitor cells (HSPCs); we injected equally treated human CFSE labeled AML cells via tail vein injection at day versus night time points and analyzed murine BMs 8 hours later for the presence of human leukemic cells. Indeed, significantly higher BM colonization by leukemic cells was observed after night vs. day time injections (0.31±0.09 vs. 0.13±0.18, p

Description: This article reports metabolic consequences of JAK2-mutant myeloproliferative neoplasms (MPNs) with a therapeutic translational impact: expression of mutant JAK2 induces abnormal metabolic activity of MPN cells, resulting in hypoglycemia, adipose tissue atrophy, and early mortality.

Description: JAK2-V617F is the most frequently recurring somatic mutation in patients with myeloproliferative neoplasm (MPN), but it can also be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the incidence of MPN. This suggests that the acquisition of the JAK2-V617F is not the rate-limiting step and other factors might be required for the expansion of the JAK2 mutated clone and initiation of MPN disease. Chronic inflammation is a hallmark of advanced MPN and is associated with progression to myelofibrosis and AML. Interleukin-1β (IL-1β) is one of the master regulators of the inflammatory state and its aberrant activity has been implicated in various pathological diseases including MPN. Here we focused on the early stages of MPN disease initiation and examined the role of IL-1β in this context. We hypothesized that IL-1β mediated inflammation may promote early expansion of the JAK2 mutant clone to reach a critical clone size capable of initiating MPN. We used a genetic approach and crossed IL-1β knockout (IL1β-/-) mice with our tamoxifen inducible SclCreER;JAK2-V617F (VF) mice, generating a triple mutant SclCreER;JAK2-V617F;IL-1β-/-(VF;IL1β-/-) line. We then transplanted two million bone marrow (BM) cells from VF and VF;IL1β-/- mice into lethally irradiated wildtype (WT) orIL1β-/- recipients. Complete blood counts monitored every 4 weeks for up to 32 weeks post transplantation showed reduced platelet, neutrophil, leukocyte and monocyte counts in mice transplanted with VF;IL1β-/-as compared to VF. Furthermore, terminal analysis at week 16 and 32 revealed reduced splenomegaly and bone marrow fibrosis in the mice receiving VF cells lacking IL1β. This experiment shows that IL1β plays an important role in MPN pathogenesis in this mouse model. To test the hypothesis that IL1β favors clonal expansion during MPN disease initiation, we performed competitive dilution assays by mixing BM cells from VF or VF;IL1β-/-mice that also co-express the GFP protein as a reporter (VF;GFP or VF;IL1β-/-;GFP) with BM cells from IL1β-/- mice in 1:100 ratio and transplanted into lethally irradiated WT recipients (Figure 1A). Successful engraftment was defined by presence of 〉1% GFP+ cells within Gr-1+ granulocytes in peripheral blood (PB) at week 18 after transplantation. In mice transplanted with VF;GFP, we found engraftment in 25 of 29 (86%) recipients whereas in mice transplanted with VF;IL1β-/-;GFP, only 18 of 29 (62%) recipients showed engraftment. Moreover, 10 of 25 (40%) mice engrafted with VF;GFP developed MPN at 24 weeks after transplantation as compared to only 2 of 18 (11%) mice engrafted with VF;IL1β-/-;GFP cells. GFP chimerism measured every 6 weeks in peripheral blood (PB) from erythroid (Ter119+), megakaryocytic (CD61+) and granulocytic lineages (Gr-1+) was significantly reduced in mice transplanted with VF;IL1β-/-;GFP compared to mice transplanted with VF;GFP cells (Figure 1A), suggesting the capacity to produce IL-1β protein by the VF cells was promoting the expansion of the clone and MPN manifestation.To define the relative contributions of hematopoietic and non-hematopoietic cell derived IL-1β in promoting MPN initiation, we performed competitive dilution assays in IL1β-/-recipients (Figure 1B). We detected engraftment in 27 of 30 (90%) IL1β-/-recipients transplanted with VF;GFP and 27 of 33 (82%) mice transplanted with VF;IL1β-/-;GFP. Furthermore, 9 of 27 (33%) mice engrafted with either VF;GFP or VF;IL1β-/-;GFP developed MPN at 24 weeks after transplantation. However GFP chimerism in Ter119, Gr-1 and CD61 was lower in mice transplanted with VF;IL1β-/-;GFP compared to mice transplanted with VF;GFP (Figure 1B). We further looked at plasma IL-1β protein levels by ELISA (Figure 1C). Interestingly, we found that IL-1β protein levels were also reduced in WT mice transplanted with VF;IL1β-/-;GFP donor cells, indicating that the non-hematopoietic WT cell cannot compensate for the deficiency of IL-1β in the VF clone. Overall, our results demonstrate that IL-1β favors early clonal expansion and show that IL-1β produced by the JAK2 mutant cells is required for optimal MPN disease initiation. Disclosures No relevant conflicts of interest to declare.

Description: The SAKK 35/10 phase 2 trial, developed by the Swiss Group for Clinical Cancer Research and the Nordic Lymphoma Group, compared the activity of rituximab vs rituximab plus lenalidomide in untreated follicular lymphoma patients in need of systemic therapy. Patients were randomized to rituximab (375 mg/m2 IV on day 1 of weeks 1-4 and repeated during weeks 12-15 in responding patients) or rituximab (same schedule) in combination with lenalidomide (15 mg orally daily for 18 weeks). Primary end point was complete response (CR)/unconfirmed CR (CRu) rate at 6 months. In total, 77 patients were allocated to rituximab monotherapy and 77 to the combination (47% poor-risk Follicular Lymphoma International Prognostic Index score in each arm). A significantly higher CR/CRu rate at 6 months was documented in the combination arm by the investigators (36%; 95% confidence interval [CI], 26%-48% vs 25%; 95% CI, 16%-36%) and confirmed by an independent response review of computed tomography scans only (61%; 95% CI, 49%-72% vs 36%; 95% CI, 26%-48%). After a median follow-up of 4 years, significantly higher 30-month CR/CRu rates and longer progression-free survival (PFS) and time to next treatment (TTNT) were observed for the combination. Overall survival (OS) rates were similar in both arms (≥90%). Toxicity grade ≥3 was more common in the combination arm (56% vs 22% of patients), mainly represented by neutropenia (23% vs 7%). Addition of lenalidomide to rituximab significantly improved CR/CRu rates, PFS, and TTNT, with expected higher, but manageable toxicity. The excellent OS in both arms suggests that chemotherapy-free strategies should be further explored. This trial was registered at www.clinicaltrials.gov as #NCT01307605.

Description: Background: The randomized phase-2 trial SAKK 35/10 was conducted by the Swiss Group for Clinical Cancer Research (SAKK) and the Nordic Lymphoma Group (NLG) to compare the activity of single-agent rituximab versus rituximab plus lenalidomide in the first-line treatment of symptomatic follicular lymphoma (FL). The results of primary endpoint (complete remission [CR/CRu] at week 23) assessment were previously reported, showing that addition of lenalidomide to rituximab results in a significantly higher CR/CRu rate at the expected cost of increased but manageable toxicity (Kimby et al. Blood 2014.124 (21):799; Zucca et al. Hematol Oncol 2015. 33(s1): 105). Here we report the first analysis of secondary endpoints, progression-free survival (PFS), time to next anti-lymphoma treatment (TTNT), CR duration, as well as CR/CRu rate at 30 months (CR30). Methods: 154 patients (pts) with grade 1 to 3a FL, untreated and in need of systemic therapy, were randomized to receive either rituximab (375mg/m2 at week 1, 2, 3, 4, 12, 13, 14 and 15) or rituximab (same schedule) plus lenalidomide (15 mg daily, from 14 days before the first until 14 days after the last rituximab administration). The sample size was calculated to allow the detection of a 20% increase of the CR/Cru rate with 90% power and type I error 0.10; a one-sided Z-test for proportions was used to compare the two arms. Treatment was discontinued in pts who did not achieve at least a 25% reduction in the sum of products of tumor diameters at week 10. Primary and secondary endpoints were defined according to the NCI international standardized criteria (Cheson et al 1999). Results: 77 pts (median age 63 years, 52% with stage IV and 47% with poor-risk FLIPI score) were allocated in the single-agent rituximab arm and 77 (median age 61 years, 48% with stage IV and 47% with poor-risk FLIPI score) in the combination arm. A higher CR/CRu rate in the combination arm was documented both by the investigator assessment (36% vs 25%) and by the independent response reviewers of CT scans (61% vs. 36%). Adverse events of grade ≥3 were more common (56% vs 22% of pts) in the combination arm, including neutropenia (23% vs 7%). At a median follow up of 3.1 years, a longer CR duration was seen for the pts in the combination arm (median not reached vs 2.3 years) as well as a longer PFS (median not reached vs. 2.3 years), these differences were not statistically significant. The CR30, recently identified as a reliable surrogate of PFS (Sargent et al. Hematol Oncol 2015. 33(s1): 166), was significantly improved by the addition of lenalidomide to rituximab (42% vs 19%, p=0.001). Moreover, TTNT was significantly longer with the combination (median not reached vs 2.1 years, p=0.02) [Figure1]. Overall survival rates at 3 years were 93% and 92%, respectively. Conclusions: The SAKK 35/10 randomized trial confirmed that lenalidomide plus rituximab is an active and feasible initial treatment for FL pts in need of therapy. Addition of lenalidomide significantly increased the CR/CRu rate at week 23 (primary endpoint) and was maintained throughout 30 months. Although the trial was not powered to detect survival differences (secondary endpoints), a significantly better TTNT and a trend towards prolonged PFS and CR duration was seen in the combination arm. The excellent overall survival in both arms suggests that chemotherapy-free strategies should be further explored. Figure 1. Time to next anti-lymphoma therapy by treatment arm Figure 1. Time to next anti-lymphoma therapy by treatment arm Disclosures Kimby: Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Honoraria for lecture at educational session; Celgene: Other: Honoraria for lecture. educational meeting; Pfizer: Other: Research grant; Roche: Other: Honoraria for lecture in educational meetings; Gilead: Honoraria, Other: honoraria for educational lecture in meeting sponsored by Gilead. Mey:roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding. Wahlin:Roche: Consultancy. Hernberg:Roche: Consultancy, Honoraria. de Nully Brown:Roche: Research Funding. Ferreri:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Zander:Bristol Myers, Celgene, Amgen, Mundipharma, Janssen-Cilag, Takeda Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Xenografts in immune suppressed mice are routinely used for in vivostudies on human acute myeloid leukemia (AML). However, a significant proportion of primary AML samples (»50-60% of cases) were engrafting in mice with particularly latency. Here, we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) accelerates the engraftment of human AML cells in NOD/SCID/IL2Rgnull(NSG) mice. Equal numbers of freshly thawed AML cells from the same donor (n=5 patients) were transplanted into sublethally irradiated gender-matched 6 to 8 weeks old mice at day or at night (n=3-5 mice per condition and patient). Transplanted mice were monitored for leukemic engraftment in peripheral blood (PB), bone marrow (BM) and other hematopoietic organs using flow cytometry and immunohistochemistry. Enhanced leukemic engraftment was observed in night vs. day transplanted mice for each AML case (Fig. A). Interestingly, in 2/5 samples, engraftment was exclusively detected with the night transplant condition. Limiting dilutions showed that night transplantation allows leukemia (Fig. B) induction from lower cell numbers. Results were confirmed in a syngeneic MLL-PTD/FLT3-ITD mouse leukemia model (Fig. C): mice analyzed at the same time-point after night versus day transplantation showed enhanced splenomegaly (401.2 mg vs. 234.5 mg, p

Description: We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and show bias for differentiation towards megakaryocytes (Mk). Mouse models of myeloproliferative neoplasms (MPN) expressing JAK2-V617F (VF) or a JAK2 exon 12 mutation (E12) displayed increased frequencies and percentages of the CD41hi versusCD41lo HSCs compared to wildtype controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from MPN patients. CD41hi HSCs produced higher numbers of Mk-colonies HSC in single cell cultures in vitro, but showed reduced long-term reconstitution potential compared to CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, while CD41lo HSCs showed higher gene expression of interferon, JAK/STAT and TNFα/NFkB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in MPN patients. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the numbers of JAK2-V617F positive HSCs in mice and patients with MPN. The shift towards the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

Description: Myeloproliferative neoplasms (MPNs) are clonal hematological malignancies, which are initiated and maintained by a pool of malignant long-term hematopoietic stem cells (LT-HSCs). A recurrent gain-of-function mutation in JAK2-V617F (VF) is present in a majority of patients with MPN. Treatment with pegylated interferon alpha (pegIFNα) is currently the only therapy known to reduce JAK2-V617F+ LT-HSCs and to induce a complete molecular remission (CMR) in a subset of patients. Long-term follow-up studies suggested, that patients with additional mutations in epigenetic modifiers, such as DNMT3A, have poorer responses to pegIFNα and are less likely to achieve CMR. We therefore hypothesized that DNMT3A loss leads to alterations in JAK2-V617F HSCs functions conferring resistance to pegIFNα treatment. To test the hypothesis we performed competitive transplantations of bone marrow cells from VF or VF;Dnmt3a-/- mice, which also co-express the GFP reporter mixed with wildtype competitor (WT) cells in 1:20 ratio (Figure 1A). After 6 weeks, recipients mice were randomized and treated for 16 weeks with mouse pegIFNα (25µg/kg; s.c.; once per week) or saline (Figure 1A). Complete blood counts and GFP chimerism monitored every 4 weeks showed, that pegIFNα was able to achieve normalization of peripheral blood parameters and reduce GFP chimerism in both, VF and VF;Dnmt3a-/- chimeric mice. In VF chimeric mice treated with pegIFNα we observed a significant reduction of splenomegaly (Figure 1B) and normalization of bone marrow MPN histopathology compared to saline, however pegIFNα treatment in double-mutant VF;Dnmt3a-/- did not improve histopathological features and had tendency to worsen splenomegaly. GFP chimerism in LT-HSCs and committed hematopoietic progenitors was reduced only in VF chimeric mice, but not in double mutant mice. Chronic activation and ensuing replication-induced DNA damage is a critical factor for the functional decline and depletion of mutant LT-HSCs upon treatment with pegIFNα. Our functional analysis shows that LT-HSCs and early progenitors from double-mutant VF;Dnmt3a-/- mice were less prone to accumulate reactive oxygen species, DNA damage and exit dormancy upon treatment with pegIFNα, compared to VF (Figure 1B). In series of secondary transplantations at increasingly limiting dilutions, bone marrow cells from VF mice treated with pegIFNα showed reduced repopulation capacity resulting in a milder MPN phenotype and lower mutant LT-HSC chimerism in the secondary recipients. In contrast, bone marrow cells from VF;Dnmt3a-/- double mutant mice treated with pegIFNα gave rise to a more aggressive disease phenotype compared to saline in secondary (Figure 1C) and also tertiary transplantations (not shown). Recipients transplanted with BM cells from VF;Dnmt3a-/- mice treated with pegIFNα also showed increased frequencies of mutant LT-HSCs compared to saline. Our results suggest that the accelerating effects of pegIFNα therapy inflicted on the LT-HSC of VF;Dnmt3a-/- double mutant mice are stably inherited upon secondary and tertiary transplantations. Overall, our results demonstrate that loss of Dnmt3a mitigate functional attrition of JAK2-V617F+ LT-HSCs upon chronic pegIFNα treatment and create vastly expanded pool of resistant LT-HSCs and point towards possible detrimental effects of pegIFNα therapy in patients with additional loss-of-function mutation in DNMT3A. Disclosures No relevant conflicts of interest to declare.