FLT3 Receptor Research Articles

VEGF-R1/Flt-1 Receptor as a Characteristic of the Angiogenic Phenotype of Uveal Melanoma

Thanks to new fundamental data, our understanding of the angiogenesis’ role and its molecular participants has changed. Molecular genetic mechanisms of activation of signaling pathways of proliferation, malignancy, suppression of the tumor cells’ apoptosis, carried out through the interaction of the VEGF molecule with its receptors, have been deciphered. Molecular genetic mechanisms of activation of signaling pathways of proliferation, malignancy, suppression of apoptosis of tumor cells, carried out through the interaction of the VEGF molecule with its receptors, have been deciphered. The doctrine of angiogenesis has changed. Angiogenesis began to be considered in the aspect of the increasing anaplasia mechanism, accelerating proliferation, the formation of a clone of stem tumor cells, highly resistant to chemotherapy and radiation therapy with a high potential for metastasis. It is time to reconsider the role of individual biological markers of angiogenesis in their suitability in predicting disease outcome and evaluating them as a potential target for targeted therapy. In this aspect, uveal melanoma (UM) as a model of an extremely aggressive malignant tumor using its angiogenic phenotype to accelerate hematogenous metastasis is of particular interest. One of the characteristics of the angiogenic phenotype is VEGF-R1/Flt-1. The purpose: to study the expression of the VEGF-R1/Flt-1 receptor as a characteristic of the angiogenic phenotype of UM in correlation with its clinical and morphological indicators and the outcome of the disease. The authors conducted a retrospective study on 98 archival paraffin blocks of the eyes of patients with UM. The following general patterns of Flt expression in UM cells are revealed: Basic expression takes place in the smallest tumor proliferates UM (T1). Moreover, overexpression (IGH-gradation III) of Flt in the nucleus (39.7 %) and cytoplasm (36.3 %) occurred in every third patient with UM in stage T1. The peak of reception for the maximum average percentage of positive cells and the average cellular IGH gradation of expression was recorded at the T2 stage, after which there was a slow decline to the T4 stage. The number of overexpressive Flt in the nucleus and cytoplasm of UM cells increased x 2 times (75.3 %) to the T2 stage. The peak of Flt nuclear expression in terms of the number of immunopositive cells was also recorded at the T2 stage. The authors concluded that VEGF-R1/Flt-1 expression is a very important characteristic of the UM angiogenic phenotype. In the vast majority of UM, there is an expression of the VEGF-R1/Flt-1 receptor in the nucleus and cytoplasm of tumor cells. The revealed correlations of VEGF-R1/Flt-1 expression with the volume and histological type of tumor, disease stage and metastasis allow them to consider Flt-1 an important indicator associated with the pathogenesis and prognosis of uveal melanoma and a potential target for targeted therapy. A prognostic adverse factor in the aspect of the prognosis of the risk of metastases should be considered the index of the ratio Flt C/Flt N ≥ 3.

Effects of P4 Antagonist RU486 on VEGF and Its Receptors' Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles.

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors’ signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles’ microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.

PARP1 PARylates and stabilizes STAT5 in FLT3-ITD acute myeloid leukemia and other STAT5-activated cancers

Signal transducer and activator of transcription 5 (STAT5) signaling plays a pathogenic role in both hematologic malignancies and solid tumors. In acute myeloid leukemia (AML), internal tandem duplications of fms-like tyrosine kinase 3 (FLT3-ITD) constitutively activate the FLT3 receptor, producing aberrant STAT5 signaling, driving cell survival and proliferation. Understanding STAT5 regulation may aid development of new treatment strategies in STAT5-activated cancers including FLT3-ITD AML. Poly ADP-ribose polymerase (PARP1), upregulated in FLT3-ITD AML, is primarily known as a DNA repair factor, but also regulates a diverse range of proteins through PARylation. Analysis of STAT5 protein sequence revealed putative PARylation sites and we demonstrate a novel PARP1 interaction and direct PARylation of STAT5 in FLT3-ITD AML. Moreover, PARP1 depletion and PARylation inhibition decreased STAT5 protein expression and activity via increased degradation, suggesting that PARP1 PARylation of STAT5 at least in part potentiates aberrant signaling by stabilizing STAT5 protein in FLT3-ITD AML. Importantly for translational significance, PARPis are cytotoxic in numerous STAT5-activated cancer cells and are synergistic with tyrosine kinase inhibitors (TKI) in both TKI-sensitive and TKI-resistant FLT3-ITD AML. Therefore, PARPi may have therapeutic benefit in STAT5-activated and therapy-resistant leukemias and solid tumors.

Abstract 14155: Exposure to sFlt1-Induced Preeclampsia Enhances the Future Vascular Response to Hypertensive Stimuli in Mice in a Smooth Muscle Mineralocorticoid Receptor Dependent Manner

Preeclampsia (PE), a syndrome of high blood pressure (BP) and renal damage in late pregnancy, is associated with increased soluble VEGF receptor (sFlt1) and survivors have increased risk of future hypertension with increased angiotensin II (AngII) and salt sensitivity. Since smooth muscle cell mineralocorticoid receptor (SMC-MR) can be activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure during pregnancy may induce a post-partum state of enhanced vascular sensitivity via SMC-MR activation. A PE model was induced by transient viral expression of sFlt1 in pregnant C57Bl6 mice. Elevated serum sFlt1, BP and glomerular endotheliosis was confirmed which all resolve post-partum. Two months later, resistance arteries from post-PE mice show no change in vasoconstriction to AngII but with increased mRNA ratio of the pro-constrictive AngII receptor-1(ATR1) to the vasodilatory ATR2. In a small cohort of women with prior PE, we confirm salt sensitivity of BP thus, postpartum mice were implanted with telemetric BP monitors and exposed to high salt or AngII (600mg/kg/day) infusion. Mice with prior PE had a significantly increased BP response to hypertensive stimuli. Microvessels from mice after PE and hypertensive stimuli had enhanced ex vivo myogenic tone and AngII vasoconstriction. To test the direct impact of sFlt1 on SMC-MR function, cultured SMC (Pac1) were transiently exposed to sFlt1 (24 hours on then off) to mimic the post-PE state. SMC-MR transcriptional activity in response to aldosterone and AngII were significantly increased after sFlt1 exposure as measured by luciferase reporter assay. SMC-MR activation and target gene expression was also enhanced after sFlt1 exposure (including AT1R/AT2R ratio). Finally, the role of SMC-MR in the post-PE phenotype was tested in vivo by PE induction in SMC-MR-KO vs MR-intact littermates. Post partum, SMC-MR-KO mice were protected from the PE-induced increase in aortic stiffness, microvascular myogenic tone and AngII vasoconstriction. sFlt1-induced PE produces a state of enhanced sensitivity to AngII that may be mediated by increased activation of SMC-MR. These data support the use of MR antagonists to mitigate the increased risk of cardiovascular disease in women exposed to PE.

The CBFβ-SMMHC/NRP1 Axis Regulates FLT3 and TGF-Beta Pathways in Inv(16) Acute Myeloid Leukemia

Signal transduction pathways regulate the proliferation and viability of acute myeloid leukemia (AML) blasts. The regulation in the expression of cytokine receptors in AML is not well understood. In this study, we investigated how the CBFβ-SMMHC fusion protein regulates expression of cytokine receptors in inv(16) AML, with focus on the co-receptor Neuropilin-1 (NRP1).Knock-down of CBFβ-SMMHC expression, utilizing shRNA transduction, induced G1 phase of cell cycle arrest and reduced the viability of inv(16) ME-1 cells in culture. Expression profile analysis of CBFβ-SMMHC knock-down cells revealed a significant repression of genes associated with transmembrane receptor protein kinase pathways, including NRP1 (-5 fold), FGFR1 (-4.2 fold) , FLT3 (-2 fold) and TGFBR2 (-1.2 fold). The expression of NRP1 was significantly upregulated in inv(16) AML cases when compared to other AML sub-types and to hematopoietic stem and progenitor cells. Functionally, NRP1 knock-down reduced the viability of ME-1 cells with a similar dynamics as when using CBFβ-SMMHC shRNAs. In addition, the proliferation of inv(16) AML cells was reduced 4.1-fold when treated with a function-blocking antibody for the FV/VIII extracellular NRP1 domain, while having no effect in non-inv(16) AML cells or when using blocking antibody for the CUB extracellular domain. Furthermore, deletion of Nrp1 by gene editing reduced the colony-forming unit capacity of primary mouse Cbfb +/MYH11 leukemic cells and extended the median leukemia latency in vivo. To identify the genes regulated by NRP1 in inv(16) AML, we analysed the transcription profile of NRP1 knock-down in ME-1 cells. Gene Set Enrichment and Pathway Analysis revealed a repression in STAT5 pathway, and in signalling receptor activity, including FLT3 (-1.8 fold) and TGFBR2 (-1.8 fold) expression, indicating that NRP1 mediates transcriptional regulation of FLT3 and TGFBR2 expression in inv(16) AML. Furthermore, the regulation of FLT3 and TGFB2 expression by CBFβ-SMMHC and by NRP1 was validated by gene editing in inv(16) AML blasts. Accordingly, NRP1 knock-down in AML cells reduced SMAD2/3 phosphorylation. The repression of RUNX1/CBFβ function, using small molecule inhibitors, in inv(16) AML cells with CBFβ-SMMHC knockdown restored NRP1 expression, suggesting that RUNX1 may repress NRP1 expression in AML cells. To evaluate if RUNX1 directly regulates NRP1 expression, we tested RUNX1 binding in the NRP1 locus of AML cells with CBFβ-SMMHC knockdown. RUNX1 binding at one of six sites with RUNX1 occupancy identified by chromatin immunoprecipitation followed by sequencing (RE5, regulatory element 5) was increased in the CBFβ-SMMHC knock-down cells. The RE5 is located 178kb upstream of the NRP1 transcription start site and it is evolutionarily conserved in vertebrates. The deletion of RE5 by gene editing (~50% editing efficiency) increased NRP1 expression 1.8-fold, suggesting that RUNX1 may repress NRP1 expression at by binding to the RE5 enhancer.Taken together, these studies demonstrate that CBFβ-SMMHC regulates expression of cytokine receptors in inv(16) AML. Specifically, it directly regulates expression of the co-receptor NRP1, which is essential for AML survival, acting (at least in part) by regulating FLT3 and TGFB pathways. DisclosuresGuzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

Anti-FLT3 CAR T Cells in Acute Myeloid Leukemia

INTRODUCTIONIn patients with AML who are eligible for intensive therapy, the goal of treatment is the achievement of complete response followed by consolidation chemotherapy (in favorable risk disease) or hematopoietic stem cell transplantation (in intermediate or adverse risk disease). Patients who do not attain this initial goal lack effective therapeutic options. Extensive experience with chimeric antigen receptor (CAR) T cells in B-ALL has shown that CART cells can deliver potent and durable antigen-specific leukemia control, and that targeting a single antigen (CD19 for B-ALL) is associated with antigen-negative relapse. In this context, we sought to expand the existing preclinical CART armamentarium in AML by developing FLT3-specific CART cells and comparing them to our existing gold standard CD123-specific CART cells. Since activating mutations in FLT3 occur commonly in AML, we reasoned that this molecule would serve as an “Achilles heel” in AML immunotherapy.METHODSNovel fully humanized anti-human FLT3 receptor single chain variable fragments (scFV) were fused to CD28 and CD137 (41BB) costimulatory molecules and the CD3zeta signaling domain and cloned into a lentiviral expression vector. Based on recently published data, we tested linker lengths ranging from 5 to 20 amino acids between the light and heavy chains of the CAR. We used a FLT3-ITD mutated AML cell line (MOLM14) expressing luciferase for in vitro function studies including an exhaustion assay. For in vivo function studies, we engrafted MOLM14 expressing luciferase into NSG mice and treated with CART-FLT3 or untransduced T cells (negative control).RESULTSAll FLT3 and CD123-specific CART cells degranulated and produced the effector cytokines IL-2, INFg, TNF and GM-CSF in an antigen-specific manner, with some variability between the different linker lengths and with some superiority of the CAR123 likely resulting from the higher expression of CD123 compared with FLT3 in this model (p < 0.0001, one way ANOVA) (Figure 1). Short-term killing assays (24 hours) revealed that all CART cells killed MOLM14 with equivalent efficiency at low effector:target ratios (Figure 2A). Since short-term killing assays likely do not replicate the physiological situation in vivo wherein CART cells encounter cancer cells repeatedly over many days, we next developed an in vitro exhaustion assay. We incubated MOLM14 cells with CAR T cells at 1:10 E:T ratio and added MOLM 14 tumor cells along with fresh media every other day. Killing was quantified every 48 hours. Interestingly, all CAR constructs showed equivalently efficient cytotoxicity from days 5-15. However, after day 15 there was progressive dysfunction and loss of cytotoxic activity. This exhaustion “stress test” revealed some superiority of the FLT3 CAR 10AA construct (p = 0.042 on day 17, two way ANOVA) (Figure 2B).NOD/SCID gamma chain KO (NSG) mice were then engrafted with 1x10 6 luciferized MOLM14 cells and treated with 0.5x10 6 CAR T cells 7 days later, randomized to treatment groups based on tumor burden. CAR T cells expansion was monitored in peripheral blood by flow cytometry. (Fig 3A). Serial BLI revealed prompt and durable leukemia remissions and survival (Figure 3B,C).CONCLUSIONSWe have developed CART-FLT3 for AML using novel human anti-FLT3 targeting domains and demonstrated preclinical efficacy similar to that of CART-123 in an AML model with substantially lower expression of FLT3 compared to CD123 (data not shown). Since inhibition of FLT3 leads to upregulation of surface FLT3 expression, future experiments will explore combinatorial FLT3 inhibition with CART-FLT3. If successful, these experiments will provide a strong rationale for a combination clinical trial in AML where leukemia control by small molecules is followed by a coup-de-grace delivered by CART cells. [Display omitted] DisclosuresSleiman: Hemogenyx Pharmaceuticals LLC: Research Funding. Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Santiago: Hemogenyx Pharmaceuticals LLC: Research Funding. Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang: Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda: Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler: Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Elastin-Like Polypeptide: VEGF-B Fusion Protein for Treatment of Preeclampsia.

[Figure: see text].

Novel β‑hydroxy ketones: Synthesis, spectroscopic characterization, molecular docking, and anticancer activity studies

In this study, a series of novel β‑hydroxy ketone derivatives 3a-o were designed, synthesized, and evaluated for their anticancer activity. The structure of these compounds were characterized by IR, 1H and 13C NMR, mass spectrometry and elemental analysis methods. All the synthesized compounds were screened for anticancer activity against MCF-7 and U87 cells. Among them, compound 3l was appeared to be the most potent compound on both cancer cells; and IC50 dose was determined as 145 µM for MCF-7 cells and 6,6 µM for U87 cells. DNA ladder and Annexin V apoptotic marker tests were used and as a result, 3l caused the initiation of apoptosis on U87 cells. VDAC protein expression increased dramatically after U87 glioblastoma brain cancer cells were treated with compound 3l Additionally, the molecular modeling of these compounds was studied in FLT3 receptor, Estrogen receptor, and PARP2 receptor for the treatment of Acute Myeloid Leukemia (AML), breast cancer, and Glioblastoma (GBM) respectively. Their binding motifs and drug-like properties were investigated, and the results are highlighted in the discussion. Based on the results, compound 3l may have a potential drug candidate profile that can reverse the drug resistance profile.

Gilteritinib-induced upregulation of S100A9 is mediated through BCL6 in acute myeloid leukemia

AbstractDrug resistance and relapse are common challenges in acute myeloid leukemia (AML), particularly in an aggressive subset bearing internal tandem duplications (ITDs) of the FLT3 receptor (FLT3-ITD+). The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet resistance to gilteritinib remains a clinical concern, and the underlying mechanisms remain incompletely understood. Using transcriptomic analyses and functional validation studies, we identified the calcium-binding proteins S100A8 and S100A9 (S100A8/A9) as contributors to gilteritinib resistance in FLT3-ITD+ AML. Exposure of FLT3-ITD+ AML cells to gilteritinib increased S100A8/A9 expression in vivo and in vitro and decreased free calcium levels, and genetic manipulation of S100A9 was associated with altered sensitivity to gilteritinib. Using a transcription factor screen, we identified the transcriptional corepressor BCL6, as a regulator of S100A9 expression and found that gilteritinib decreased BCL6 binding to the S100A9 promoter, thereby increasing S100A9 expression. Furthermore, pharmacological inhibition of BCL6 accelerated the growth rate of gilteritinib-resistant FLT3-ITD+ AML cells, suggesting that S100A9 is a functional target of BCL6. These findings shed light on mechanisms of resistance to gilteritinib through regulation of a target that can be therapeutically exploited to enhance the antileukemic effects of gilteritinib.

PIGF and Flt-1 on the surface of macrophages induces the production of TGF-β1 by polarized tumor-associated macrophages to promote lung cancer angiogenesis

BackgroundThe interaction between tumor cells and tumor microenvironment is a necessary condition for promoting the metastasis of malignant tumors. MethodsTwo different transwell culture systems were interfered with by recombinant factor placental growth factor (re-PIGF) and the re-PIGF + transforming growth factor-β1 (TGF-β1)-neutralizing antibody (anti-TGF-β1). We performed immunofluorescence, flow cytometry and enzyme linked immunosorbent assay (ELISA) to analyze the expression of PIGF, fms-like tyrosine kinase-1 (Flt-1), macrophage marker F4/80 +, macrophage M2 marker CD163+ and TGF-β1 in vitro. Meanwhile, cell viability assay and optical microscope assay were conducted to explore the cell viability and vascularization ability of human umbilical vein endothelial cells (HUVECs). ResultsRe-PIGF increased the expression of PIGF in A549 cells and the expression of Flt-1 in BM-Mac cells, and significantly enhanced the ability of bone marrow-derived macrophages (BM-Mac) to transform into macrophages. At the same time, re-PIGF increased the expression of cytokine TGF-β1 in A549 cells/BM-Mac transwell culture system. On the contrary, re-PIGF + anti-TGF-β1 inhibited the expression of Flt-1 in BM-Mac cells and inhibited the ability of BM-Mac cells to transform into macrophages. Finally, re-PIGF + anti-TGF-β1 reduced the cell viability and angiogenesis of HUVECs. ConclusionThe surface molecule PIGF of lung cancer cells could bind to the receptor Flt-1 on the surface of macrophages, thereby increasing the production of TGF-β1, and ultimately promoting the formation of angiogenesis in lung cancer.

AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

Poster: AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

Poster: AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

VEGF Maintains Maternal Vascular Space Homeostasis in the Mouse Placenta through Modulation of Trophoblast Giant Cell Functions.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that acts primarily on endothelial cells, but numerous studies suggest that VEGF also acts on non-endothelial cells, including trophoblast cells. Inhibition of VEGF signaling by excess production of the endogenous soluble VEGF receptor sFlt1 in trophoblast cells has been implicated in several pregnancy complications. Our previous studies and other reports have shown that VEGF directly regulates placental vascular development and functions and that excess VEGF production adversely affects placental vascular development. Trophoblast giant cells (TGCs) line the maternal side of the placental vasculature in mice and function like endothelial cells. In this study, we specifically examined the effect of excess VEGF signaling on TGC development associated with defective placental vascular development using two mouse models an endometrial VEGF overexpression model and a placenta-specific sFlt1 knockdown model. Placentas of endometrial VEGF-overexpressing dams at embryonic days (E) 11.5 and 14.5 showed dramatic enlargement of the venous maternal spaces in junctional zones. The size and number of the parietal TGCs that line these venous spaces in the placenta were also significantly increased. Although junctional zone venous blood spaces from control and VEGF-overexpressing dams were not markedly different in size at E17.5, the number and size of P-TGCs were both significantly increased in the placentas from VEGF-overexpressing dams. In sFlt1 knockdown placentas, however, there was a significant increase in the size of the sinusoidal TGC-lined, alkaline phosphatase-positive maternal blood spaces in the labyrinth. These results suggest that VEGF signaling plays an important role in maintaining the homeostasis of the maternal vascular space in the mouse placenta through modulation of TGC development and differentiation, similar to the effect of VEGF on endothelial cells in other vascular beds.

Abstract 1428: PARP inhibitor resensitizes TKI-resistant AML to TKI

Abstract Internal tandem duplications within the juxtamembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) are present in approximately 25% of patients with acute myeloid leukemia (AML) and is an independent predictor of poor prognosis. FLT3-ITD is a constitutively active form of the receptor and aberrantly signals through signal transducer and activator of transcription (STAT) 5 which feeds-back to stabilize FLT3 through the downstream effector, Pim-1 kinase. FLT3-specific tyrosine kinase inhibitors (TKI) have clinical effects in FLT3-ITD AML but responses are limited due to the development of resistance through various means, including point mutations in the FLT3 tyrosine kinase domain commonly at D835 in the activation loop and F691 in the gate-keeper residue. Novel therapeutic strategies are therefore required for overcoming TKI-resistance. Poly (ADP-ribose) polymerase (PARP) 1 functions by poly-ADP-ribosylating (PAR) itself and other proteins primarily to catalyze DNA repair, however, PARP can also regulate the activity and function of proteins involved in other various processes. Notably, PARP inhibitors (PARPi) are novel cancer therapeutics that induce synthetic lethality in cancers with BRCA mutations. Recent studies also show that concurrent treatment of PARPi and TKI can effectively induce synthetic lethality in BRCA-proficient TKI-sensitive FLT3-ITD AML. However, the effects of PARPi in TKI-resistant disease has yet to be explored. We previously reported elevated reactive oxygen species (ROS) in FLT3-ITD AML leading to upregulated PARP1 and we now show that, compared to parental TKI-sensitive Molm14 cells, D835Y and F691L point-mutated TKI-resistant forms have significantly increased ROS production, and subsequently increased DNA damage and PARP1 protein expression. Interestingly, TKI-resistant cells also have elevated phospho-STAT5 (pSTAT5) and subsequent CRISPR-Cas9 knockout of PARP1 leads to reduction in pSTAT5 as well as the downstream targets, Pim-1 and FLT3. Significantly, STAT5 protein was found to immunoprecipitate with PAR and PARP1, suggesting for the first time that PARP1 may play a direct role in regulating STAT5. Finally, we have found that low doses (5-50nM) of the potent PARPi, Talazoparib (Tal), in combination with low dose (10nM) of the TKIs, Quizartinib (Quiz) or Gilteritinib (Gilt), is synergistically lethal in the otherwise TKI-resistant cells. Additionally, sequential treatment of TKI-resistant cells with Tal (72hr) prior to treatment with Quiz (72hr) or Gilt (72hr) was still synergistic whereas the reverse is not. This suggests a novel model whereby Tal may resensitize cells to TKI by decreasing PARP1 effects on pSTAT5 activity which feeds back to reduce FLT3 receptor activation. Therefore, this combination has potential for treatment of TKI-resistant AML and studies are underway to translate these findings with the intent of developing an effective therapy for patients with this disease. Citation Format: Anna J. Dellomo, Rena G. Lapidus, Mariusz Karbowski, Maria R. Baer, Tami J. Kingsbury, Feyruz V. Rassool. PARP inhibitor resensitizes TKI-resistant AML to TKI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1428.

Abrogating Treatment Resistance By Mir-451 in FLT3-ITD-Positive Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a fatal malignant hematologic disorder. Although many patients with AML initially respond to standard chemotherapy and achieve complete remission (CR), the majority of patients relapse due to the development and clonal expansion of chemoresistant subclones. Albeit being a heterogenous disease, individual mutations are still scarcely considered in the current treatment regimen.Approximately 30% of AML patients exhibit gain-of-function mutations in the FMS-like tyrosine kinase 3 receptor (FLT3-receptor) gene. Here, an internal tandem duplication (ITD) in the juxtamembrane region of the receptor is among the most frequently observed mutations. This usually entails a constitutive activation of FLT3 receptor downstream signaling pathways, leading to differential gene expression patterns and aberrant cell growth. Clinically, FLT3-ITD mutations confer adverse prognosis. Thus, many promising FLT3 signaling inhibitor-based therapies are currently utilized to improve patient survival. However, the phenomenon of treatment resistant subclones persists and constantly calls for new angles of treatment.MicroRNAs, a group of small non-coding RNAs, regulate gene expression on a posttranscriptional level by a complementary binding to 3'UTRs of protein-coding mRNAs. They have been shown to influence healthy and malignant hematopoiesis and to predict treatment response in cancer. In addition, several candidates have been connected to treatment resistance or sensitivity in AML.Within this study, we hypothesized that microRNAs are biological important players in the adverse treatment response and the development of chemoresistance in FLT3-ITD associated AML.Initially, we were interested in the microRNA expression pattern in FLT3-ITD associated AML. Therefore, we performed a next generation sequencing screen in bone marrow mononuclear cells from healthy donors, AML patients with FLT3-wildtype and AML patients with FLT3-ITD mutation. Here, we identified miR-451 to be significantly lower expressed in AML compared to healthy individuals. Furthermore, it was nearly absent in FLT3-ITD associated AML. In addition, we compared our results to the AML patient cohort of “The Cancer Genome Atlas” (TCGA) and found a similar miR-451 expression distribution. In agreement with this, miR-451 expression increased during differentiation of primary human and mouse hematopoietic cells. Moreover, transduction of U937 as well as 32D cells with FLT3-ITD led to a decrease in miR-451 expression.Downregulation of miR-451 has been shown for various cancerous diseases to correlate with an increased cancer persistence as well as recurrence, as one established target of miR-451 is multi drug resistance protein 1 (MDR1). MDR1 is an ATP dependent efflux pump that mediates chemoresistance by transporting drugs out of the cell. Today, only limited data are available on the miR-451-MDR1 axis in FLT3-ITD associated AML. Thus, we hypothesized that low miR-451 expression and high MDR1 activity is associated with treatment resistance.Therefore, we showed an inverse correlation of miR-451 expression and MDR1 protein levels in various leukemic cells lines. Furthermore, we observed that a repression of FLT3-ITD signaling by both Quizartinib and Gilteritinib causes a miR-451 expression increment and a subsequent reduction of MDR1 protein levels. By luciferase activity assay, we could demonstrate a direct binding of miR-451 to the 3'UTR of MDR1. In order to investigate whether miR-451 alone is able to abrogate treatment resistance, we overexpressed miR-451 in both normal MV4;11 and treatment resistant MV4;11 cells (MV4;11-R). Subsequently, MDR1 protein levels were decreased and cells exhibited an enhanced sensitivity towards both FLT3-ITD inhibitor and chemotherapeutic agent-based treatment. Moreover, exogenous miR-451 was able to restore drug sensitivity in MV4;11-R cells comparable to that of normal MV4;11 cells. As proof of principle, MDR1 inhibitor Tariquidar mimicked miR-451-mediated effects and sensitized MV4;11-R cells to FLT3-ITD inhibitor Quizartinib.In conclusion, we identified a novel mechanism by which FLT3-ITD is able to alter treatment response and confer a poor prognosis. Hence, not only inhibiting FLT3-ITD but mimicking the effects of negatively regulated downstream targets such as miR-451 presents as promising new avenue for FLT3-ITD AML treatment. DisclosuresThiede:Bayer: Consultancy, Speakers Bureau; Roche: Consultancy; Novartis: Consultancy, Speakers Bureau; Agendix: Employment.

Comparison of the Profile of the AML Drug Midostaurin (Rydapt®) As a Kinase Inhibitor with Those of Its Predominant Primary Human Metabolites

Background: The multi-targeted kinase inhibitor midostaurin (M) has single agent activity in internal tandem duplication and tyrosine kinase domain mutant FLT3 acute myeloid leukemia (AML). When added to standard chemotherapy M (50 mg, BID) significantly improved both event-free and overall survival in AML patients harboring activating FLT3 mutations (Stone RM, NEJM 2017). In humans, M is converted into 3 primary metabolites: the 10-O-demethylated compound CGP62221 and two epimeric C3-hydroxylated compounds, e1 and e2 (Fig 1). Following the administration of a single 50 mg dose of [14C]-M, the plasma AUC0-96hr of M, CGP62221, e1 and e2 were 18.0, 22.5, 4.12 and 25.8 µg Eq*h/mL respectively (He H, Drug Metab Dispos 2017) and following 50 mg BID the steady-state plasma trough levels of M, CGP62221 and e2 were 0.82, 1.48 and 6.73 µM (Wang Y, J Clin Pharmacol 2008), indicating substantial accumulation of the latter metabolite. Here we compare the kinase profile of M with those of its predominant metabolites.Methods: Reaction of M with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone afforded a mixture of oxidation products. After purification by chromatography (silica gel) and recrysallisation, X-ray crystallography of the more lipophilic epimer (VE12.094; m.p. 298-301°C) confirmed it to have (S)-stereochemistry (Fig 1). Comparison of this epimer with the products from cryopreserved human hepatocyte (BioreclamationIVT, Baltimore, MD, USA)-mediated metabolism of M showed it to be identical (ultra high performance liquid chromatography - mass spectrometry) to e1; the corresponding 3-(R)-epimer (e2) could not be purified. CGP58546 (Hoehn P, J Antibiot 1995) was treated with benzoic anhydride to give CGP62221 (purified by chromatography; m.p. 246°C). The profiles of these compounds as kinase inhibitors were determined using recombinant enzymes (13 lipid and 320 protein kinases) in radiometric transphosphorylation assays (ProQinase GmbH, Freiburg, Germany) and where >50% inhibition was observed at 10 µM, concentrations required to inhibit activity by 50% (IC50 value) were calculated from the dose-response curves.Results: All four compounds showed inhibition of both serine-threonine and tyrosine protein kinases, consistent with their binding in a type-1 ATP-competitive binding mode. M and CGP62221 showed both a similar pattern of selectivity against 320 protein kinases, as well as similar degrees of potency, although CGP62221 (the most potent inhibitor of PRKG2) was less active against LCK (Tables 1 and 2). The presence of a hydroxyl-group in the pyrrole-ring (e1 and e2) generally reduced potency resulting in increased selectivity towards VEGFR2 (none of the compounds evaluated inhibited VEGFR1 by >50% at 10 µM). PDK1 was the only kinase substantially more sensitive to e2 compared with e1 (>3-fold). None of the compounds substantially (>50%) inhibited the activity of any lipid kinase, nor of AKT, nor of any cyclin-dependent protein kinase at 10 µM.Conclusion: The disparate effects of FLT3-targeting drugs in AML clinical trials has been commented upon (Stone RM, Blood 2017). However, the efficacy and side-effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent, but often comprise of complex cooperative effects between the properties of the parent and active metabolites. AML is a heterogeneous malignancy and activating mutations in the FLT3 and KIT receptor kinases are particularly implicated among the mutations driving the malignant phenotype (Patel JP, NEJM 2012). In the case of M, the major circulating metabolites are multi-targeted protein kinase inhibitors with activity against mutant forms of FLT3 and KIT, as well as several kinases involved in their signaling pathways or implicated in AML via stromal support, such as IGF1R (Chapuis N, Haematologica 2010), JAK (Furqan M, Biomarker Res 2013), LYN (Robinson LJ, Exp Hematol 2005), PDK1 (Zabkiewicz J, Haematologica 2014), RET (Rudat S, ASH 2016), SRC (Leischner H, Blood 2012) and SYK (Puissant A, Cancer Cell 2014). Notably, CGP62221, e1 and e2, which are major circulating components following chronic dosing of M, exhibited substantial kinase activity at physiologically relevant doses. A complex interplay between the kinase activities of M and its metabolites may contribute to the efficacy of M in AML and help engender the distinctive effects of M compared to other FLT3 inhibitors in this malignancy. [Display omitted] DisclosuresManley:Novartis International: Employment. Caravatti:Novartis International: Employment. Furet:Novartis International: Employment. Roesel:Novartis International AG: Employment. Tran:Novartis International: Employment. Wagner:Novartis Pharmaceuticals: Employment.

CG '806, a Highly Potent First-in-Class Pan-FLT3/BTK Inhibitor, Targets Multiple Clusters of Kinases Operative in Diverse Subtypes of Acute Myeloid Leukemia

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/ Bruton's Tyrosine Kinase (BTK) multi-kinase inhibitor. This small molecule was designed to inhibit the wild type (WT) and C481S mutant forms of BTK, as well as the WT and mutant forms (including the internal tandem duplication, ITD) of the FLT3 receptor tyrosine kinase. Previously (2017 AACR Hematologic Malignancies abstract # 25 and #44), CG'806 was shown to have nM potency against FLT3 mutation-driven (including ITD and point mutations in tyrosine kinase domain) human acute myeloid leukemia (AML) primary patient cells, and in cultured human (eg. MV4-11 and MOLM-13) and mouse (Ba/F3) AML cell lines. CG'806 inhibited FLT3 signaling, induced apoptosis, and eradicated MV4-11 tumors with no observed toxicity in murine xenograft models. Because AML cells demonstrate extreme heterogeneity in gene expression and because FLT3 mutations account for no more than 30% of AML patients, the effect of CG'806 was further evaluated in AML cells without FLT3 mutations. Immunoblotting demonstrated that CG'806 potently decreased phospho-BTK, phospho-aurora kinases A/B/C and phospho-H3S10 without affecting FLT3 activity in AML cells having low/normal expression levels of homozygous WT FLT3 (eg. KG-1 and NOMO-1). Measurement of BrdU incorporation by flow cytometry demonstrated that CG'806 induced G2/M cell cycle arrest or polyploidy and subsequent apoptosis in FLT3 WT AML cell lines. Interestingly, CG'806 was found to induce G0/G1 cell cycle arrest but not polyploidy in FLT3-ITD driven MV4-11 and MOLM-13 cells with no effect on activity of BTK or aurora kinases at concentrations that completely inhibited FLT3 signaling (FLT3/STAT5/ERK). These findings indicate that the molecular mechanism of cell killing by CG'806 is context-dependent and varies in different AML subtypes. Evaluation of enzyme inhibitory activity (IC50) against 583 kinase enzymes, as well as binding affinities (Kd) with 483 kinase proteins, revealed pM to low nM IC50 and Kd values for CG'806 against the FLT3, BTK, AURK, c-Src, c-Met, c-Ret, DDR, TRK and STE/MAPK families of kinases. The ability of CG'806 to target multiple families or clusters of kinases is responsible for its broad efficacy in different AML subtypes driven by different genomic aberrations. Together, these findings illustrate the ability of CG'806 to inhibit entire FLT3 family of kinases, BTK and the AURK families and pathways that are operative in AML, thereby supporting the development of CG'806 for treatment of patients with FLT3-ITD and other subpopulations of patients with AML. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

A Unique N-Phenylbenzamide Inhibitor Targeting Non-Receptor Tyrosine Kinases Potently Suppresses AML Cell Growth In Vitro and In Vivo

Acute myelogenous leukemia (AML) is a hematologic cancer correlated with advancing age and poor prognosis. While diverse genetic changes are associated with AML, activating mutations in the Flt3 receptor tyrosine kinase are associated with nearly one-third of cases and include internal tandem duplication (ITD) of the regulatory juxtamembrane region. Additionally, recent studies have implicated several non-receptor tyrosine kinases in AML oncogenic signaling, including Fes, Syk and the myeloid Src-family members Fgr, Hck and Lyn. While selective Flt3 kinase inhibitors have been developed for clinical use, they have not produced durable responses, highlighting the need for new therapeutic approaches. In this study, we investigated the therapeutic potential of a multi-targeted tyrosine kinase inhibitor, TL02-59, which is based on an N-phenylbenzamide scaffold. Kinome-wide target specificity profiling (KINOMEscan) demonstrated a relatively narrow overall specificity profile for TL02-59, with only 23 potential targets out of 456 kinases when tested at a concentration of 1 µM. To confirm the results of the kinome-wide profiling against AML-associated kinases, we tested TL02-59 using in vitro kinase assays with recombinant Fes, Syk, Fgr, Hck, and Lyn as well as wild-type and ITD forms of the Flt3 kinase domain. TL02-59 displayed remarkable potency against the myeloid Src-family kinases Fgr and Lyn, with IC50values in the 100 pM range. TL02-59 also inhibited Fes, Syk, Hck and both forms of Flt3 in vitro, albeit with IC50 values in the 150-400 nM range. We then tested TL02-59 for growth-suppressive activity against Flt3-ITD+ AML cell lines, MV4-11 and MOLM-14, and the Flt3 wild-type cell line THP-1. Remarkably, TL02-59 inhibited the proliferation of MV4-11 cells with an IC50 value of 780 pM, with slightly lower potency against the MOLM-14 cells (IC50 = 3.5 nM). Quantitative real-time RT-PCR (qPCR) and immunoblot analysis confirmed that the AML-associated TL02-59 target kinases identified in vitro, including Fgr and Lyn, are expressed and active in MV4-11 and MOLM-14 cells. In contrast, TL02-59 had no effect on THP-1 cell growth up to 1 µM, consistent with transformation of these cells by kinase-independent mechanisms. We next examined the growth suppressive activity of TL02-59 in 26 bone marrow samples from AML patients, half of which expressed Flt3-ITD and half of which expressed wild-type Flt3. Twenty of the primary AML samples responded to TL02-59 treatment with growth inhibitory IC50 values in the nM range. We then determined the relative expression levels of all possible TL02-59 target kinases from the KINOMEscan analysis in these primary AML samples by qPCR. We discovered a striking correlation between TL02-59 sensitivity and expression levels of Fgr, Hck, and Lyn, supporting the idea that these Src-family members are the primary targets for TL02-59 in AML. In contrast, no correlation was observed with Flt3 expression, consistent with the observation that the four most sensitive patient samples were wild-type for Flt3. To test the efficacy of TL02-59 in vivo , we used immunocompromised (NSG) mice engrafted with MV4-11 AML cells as a model system. Two weeks after MV4-11 cell engraftment, mice were treated once daily with TL02-59 at 1 or 10 mg/kg by oral gavage for a total of 21 days. The mice were then sacrificed and evaluated for MV4-11 cells in their peripheral blood, spleen, and bone marrow by flow cytometry and IHC. At 1 mg/kg, TL02-59 reduced MV4-11 engraftment in the bone marrow by 20% and spleen engraftment by half, while suppressing the number of leukemia cells in the peripheral blood by 70% compared to vehicle control animals. At 10 mg/kg, TL02-59 reduced bone marrow AML cells by 70% and completely eliminated leukemic cells from the spleen and peripheral blood. Overall, these results demonstrate that TL02-59 is an effective inhibitor of AML cell growth in vitro and in vivo with a mechanism of action independent of Flt3 mutational status. The primary kinase targets for this inhibitor appear to be myeloid Src-family kinases, including Fgr, Lyn and Hck. DisclosuresNo relevant conflicts of interest to declare.

Functional Evaluation and Clinical Implications of FLT3 J uxtamembrane Domain Short Variants in Pediatric AML

Introduction: Somatic FLT3 activating mutations are common in AML and occur as internal tandem duplications (FLT3 /ITD) within the juxtamembrane domain (JMD) or in the activating loop of the tyrosine kinase domain (FLT3 /ALM). There is considerable variability in the length of the ITD, varying from 6 to >300 bp. We, and others, have previously demonstrated that ITD length is associated with outcome, where those with longer ITDs based on median ITD length (>48bp) had adverse outcome. However, the underlying etiology of this association has not been established. We hypothesized that longer ITDs are more likely to disrupt the auto-inhibitory properties of the JMD, leading to enhanced receptor phosphorylation and autonomous activation. We aimed to define a biologic threshold for ITD-associated relapse risk based on the length of ITD that causes maximal activation. We also evaluated the functional properties of newly identified single nucleotide variants (SNVs) within the FLT3 JMD.Methods: To evaluate the impact of FLT3 /ITD length on receptor auto-phosphorylation, FLT3 /ITD clones with a variety of ITD lengths (6, 12, 18, 24, 30, 36, 42, 48, 54 bps) were engineered and expressed in HEK293 cells. Semi-quantitative immunoblotting was used to evaluate the level of phosphorylated FLT3 (pFLT3), and comparison of pFLT3 to non-pFLT3 was calculated and presented as fold change (FC) of pFLT3:FLT3 for the different ITD lengths. We also assessed the functionality of newly discovered JMDSNVs in a similar manner, and further evaluated their response to FLT3 inhibitors. For evaluation of the clinical impact of ITD length, of the 1,022 patients treated on AAML0531, FLT3 /ITD data including ITD length as determined by fragment length analysis and allelic ratio was available from 144 patients and was used for clinical correlation with ITD length.Results: Initial evaluation of the activation potential of the different ITD lengths showed a correlation between ITD length and FLT3 phosphorylation (Fig 1A). A modest autonomous FLT3 phosphorylation was observed at 6 and 12 bp, with incremental increase in 18bp and 24bp. Maximal pFLT3 of FC was observed at 30 bp, with no significant increases detected with longer ITDs. Therefore, a biologic threshold of 30 bp was used to evaluate the clinical outcomes of patients with short ITDs (S-ITD; <30 bps) and long ITDs (L-ITD; ≥30 bps). Among the 144 FLT3 /ITD+ patients, n=37 had S-ITDs and n=107 had L-ITDs. Overall, based on cooperating cytogenetic and molecular features, a higher number of patients with S-ITDs were considered as low risk as compared to L-ITDs (36% vs. 13%; p=0.003), with a higher incidence of t(8;21) in the S-ITD cohort. There was no correlation observed between ITD length and allelic ratio (Pearson correlation r=0.54). Evaluation of clinical outcome based on ITD length as defined above demonstrated that those with L-ITD had a 5-year overall survival of 47±13% compared to 83±16% in those with S-ITD (p=0.004; Fig1B).We additionally evaluated the functional significance of newly discovered FLT3 SNVs in the JMD. A total of 20 unique mutations affecting 13 distinct codons were identified. We evaluated the effect of each amino acid variant on FLT3 activation as measured by aberrant pFLT3. Six of the 20 mutations resulted in varying degrees of autonomous pFLT3 (E573D, L576R, V592A, F594Y, F594C, Y559C). Notably, the V592A mutation resulted in potent autonomous receptor activation, with a pFLT3 level greater than that observed the D835 FLT3/ ALM mutation. All variants resulting in autonomous pFLT3 were found to be highly sensitive to the FLT3 inhibitor crenolanib, with IC50s for inhibition of pFLT3 ranging between 1.3-5.6 nMConclusion: The JMD is a critical region of FLT3, and disruption through ITDs or SNVs can result in profound dysregulation of the receptor and downstream signaling, thus serving as oncogenic events. We show that longer ITDs result in more potent FLT3 receptor activation and are associated with inferior clinical outcome. FLT3 /ITD and ALMs have recently been shown to be excellent targets for therapeutic intervention with FLT3 inhibitors. Further investigation on the use FLT3 inhibitor therapy should focus on those patients who may derive the most therapeutic benefit from these agents, which may include those with highly damaging JMD variants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Proteogenomic Landscape of High Hyperdiploid and ETV6/RUNX1-Positive Childhood Acute Lymphoblastic Leukemia

Introduction. High hyperdiploidy (51-67 chromosomes) and ETV6/RUNX1 -fusion are the two most common primary genetic events in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), together found in more than half of the cases. In this study, we performed a proteogenomic analysis of these two subtypes, with the aim of understanding the interplay between somatic genomic aberrations, in particular copy number variants (CNVs), and gene and protein expression in leukemogenesis.Materials and methods. Eighteen high hyperdiploid and nine ETV6/RUNX1 -postive childhood BCP-ALL cases were subjected to whole exome or whole genome sequencing, SNP-array analysis, RNA-sequencing, and proteome analyses using high-resolution isoelectric focusing liquid chromatography mass spectrometry. Furthermore, SNP array analysis and RNA-sequencing data for an additional 29 high hyperdiploid and 31 ETV6/RUNX1 -positive cases were analyzed.Results. Expression data for 12,314 genes and 8,480 proteins were obtained, with 8,224 genes/proteins overlapping between the datasets. There was a clear linear relationship between the fold changes on the transcript and protein levels for high hyperdiploid cases over ETV6/RUNX1 -positive cases (Spearman correlation coefficient=0.62). For individual gene/protein pairs, genes and proteins involved in specialized functions such as hematopoiesis and amino acid metabolism showed high correlations, indicating a general regulation on the RNA level, whereas genes and proteins involved in basic cell functions such as the spliceosome, the ribosome, and oxidative phosphorylation showed lower correlations. Unsupervised principal component analyses showed separate clustering of high hyperdiploid and ETV6/RUNX1 -positive cases on both the RNA and protein levels. Dosage effects related to the chromosomal gains in high hyperdiploid ALL were clearly apparent, affecting genes both in cis (i.e., loci in the CNV) as well as in trans (i.e., loci outside the CNV). These effects were lower on the protein level than on the RNA level, showing additional layers of control for protein expression. A total of 2,627 genes and 1,098 proteins were differentially expressed between high hyperdiploid and ETV6/RUNX1 -positive BCP-ALL. Alternative splicing events were also investigated and 787 differentially expressed isoforms were identified. Allele-specific expression (ASE) analysis was performed to investigate the cis-effect of gene expression and 18 ASE events were observed in high hyperdiploid cases. Enrichment analyses on the whole gene/protein dataset showed upregulation of translation and proteasome-associated genes and proteins in high hyperdiploid cases, likely associated with an increased protein turn-over and possibly associated with an aneuploidy stress response. Overexpression of the cytokine receptors FLT3 and CRLF2 was also seen and the cytokine receptor interaction pathway was enriched. RAG1/RAG2-associated somatic recombination was enriched in ETV6/RUNX1 -positive BCP-ALL, in line with previous studies showing frequent activation of this pathway. Furthermore, pathways related to chromatin modifications and chromosome structure were markedly downregulated in high hyperdiploid BCP-ALL, suggesting a general deregulation of transcriptional control. In particular, low levels of CTCF and the cohesin complex, which are important regulators of chromatin structure, were seen in both gene and protein expression data.Conclusions. In this study, we have characterized the proteogenomic landscape of the two most common forms of childhood ALL. Our data provides new insight into pathogenesis of high hyperdiploid and ETV6/RUNX1 -postive BCP-ALL and may be used to identify novel targets for treatment. DisclosuresNo relevant conflicts of interest to declare.