scholarly journals Proteomics and Phospho-Proteomics Reveal Predictive Signatures of Response and Mechanisms of Resistance to Midostaurin Plus Chemotherapy in FLT3 Mutant Positive Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3462-3462
Author(s):  
Luis Miguel Veiga Veiga Nobre ◽  
Salvatore Federico Pedicona ◽  
Arran Dokal ◽  
Andrea Arruda ◽  
Ryan Smith ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Survival Time ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Geometric Mean ◽  
Post Treatment ◽  
Mechanisms Of Resistance ◽  
Early Relapse ◽  
Acute Myeloid

Abstract Background: Midostaurin is approved for FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML), however efficacy has also been observed in a subpopulation of FLT3 mutant-negative AML, suggesting that FLT3 mutation is not the only determinant conferring midostaurin sensitivity. Casado et al previously described phosphoprotein signatures significantly associated with ex vivo responses to midostaurin in primary AML blasts (Casado et al Leukaemia 2018). In the current study, we tested whether our signatures could group FLT3+ patients based on clinical responses to midostaurin plus chemotherapy. Methods: FLT3+ bone marrow (BM) and peripheral blood (PB) specimens were collected at diagnosis, post-treatment and relapse (n=54 cases) from the Leukemia Tissue Bank at Princess Margaret Cancer Centre. All patients in this study were treated with standard chemotherapy plus midostaurin. Protein/phosphoprotein-signatures for BM and PB samples were analysed independently. Case-studies with multiple post-treatment time-points or relapse events following second line treatments were also analysed. Peptides (proteomics) and enriched phosphopeptides (phosphoproteomics) were quantified using liquid chromatography - tandem mass spectrometry. A classification machine learning (ML) algorithm was trained to group patients based on response to treatment as a function of protein/phosphoprotein-signature status. Other features (e.g. genetic mutations, HSC-transplant) were also analysed. Differential survival analysis between patient groups was carried out with Kaplan-Meier and Log Rank test methods. Pathways upregulated in post-treatment or relapse specimens, particularly from those cases that responded poorly to chemo + midostaurin (i.e. early relapse / refractory disease) were investigated using enrichment statistical methods including kinase-substrate enrichment analysis (KSEA) and gene ontology analysis and identified as potential mechanisms of resistance. Statistical significance of enrichment was determined using parametric methods and p-values adjusted for multiple testing using the Benjamini-Hochberg method. Results: ML models were developed based on the ex-vivo phosphoproteomics signatures described in the Casado et al study, from which we trained a predictive model (model 1). Patients positive for model 1 exhibited a survival probability of 243 weeks, compared to 126 weeks in signature negative patients (averages by geometric mean, Log Rank p = 9.88e-05). As the patients in the current study received chemotherapy, in addition to midostaurin, we identified a new phosphoproteomic signature consisting of 26 phospho-sites which partially overlapped with the ex-vivo signature. Patients positive for this new phosphoproteomic signature showed a markedly longer survival time than negative patients (269 vs 76 weeks, Log Rank p = 1.30e-05 for PB and 241 vs 56, Log Rank p = 2.13e-09 for BM specimens, Table). A proteomic signature was also identified in the current study. Positive patients showed a longer survival time than negative patients (330 vs 173 weeks, Log Rank p = 5.0e-04 for PB and 460 vs 156, Log Rank p = 5.2e-06 for BM specimens, Table), however this was less differentiating than the phosphoproteomic signature. Pathways upregulated in post-treatment or relapse specimens from early relapse or refractory cases were associated with molecular functions such as cell proliferation, anti-apoptosis, non-homologous end-joining, transcriptional regulation, spliceosome and cytoskeleton remodelling. Conclusions: We have identified protein and phosphoprotein signatures with the potential to further stratify AML for midostaurin treatment. Phosphoproteomic signatures differentiated according to response better than the proteomic signatures. Pathways upregulated in relapse/refractory cases may have a role in resistance and this will be determined in follow up studies. Analysis will also be performed on FLT3 mutant-negative cases to validate the signatures and elucidate mechanisms of resistance in this group. Disclosures Veiga Nobre: Kinomica Ltd.: Current Employment. Minden: Astellas: Consultancy. Gribben: Janssen: Honoraria, Research Funding; AZ: Honoraria, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Gilead/Kite: Honoraria; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutics: Honoraria. Britton: Kinomica Ltd.: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Large Scale Ex Vivo Expansion of Γδ T Cells Using Artificial Antigen Presenting Cells for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Justin C. Boucher ◽  
Bin Yu ◽  
Gongbo Li ◽  
Bishwas Shrestha ◽  
Jeffrey E. Lancet ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Large Scale ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Artificial Antigen ◽  
Antigen Presenting ◽  
Acute Myeloid

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a GMP-compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, >90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

FLT3 Inhibitors for the Treatment of Acute Myeloid Leukemia: An Evaluation of Efficacy of Target Inhibition and Relationship to Disease Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4940-4940 ◽  
Author(s):  
Adam Cloe ◽  
Richard A. Larson ◽  
Jason X. Cheng
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Poor Prognosis ◽  
Post Treatment ◽  
Flt3 Inhibitors ◽  
Before And After ◽  
Pre Treatment ◽  
Acute Myeloid

Abstract Introduction FLT3 is a receptor tyrosine kinase that plays a role in hematopoietic stem/progenitor cell proliferation and survival and is frequently found to be mutated in patients with acute myeloid leukemia (AML). Mutations that lead to constitutive activation of FLT3 (such as internal tandem duplications of the juxtamembrane domain or point mutations involving the kinase domain) are associated with a poor prognosis. This poor prognosis is in part due to an increased relapse rate after allogeneic hematopoietic cell transplant (HCT). Several compounds that inhibit the activity of FLT3 in vitro, including ASP2215, midostaurin and quizartinib, are now being studied in clinical trials for the treatment of AML. Inhibition of the formation of phospho-FLT3 has been correlated with clinical anti-leukemia effects and remissions. Methods In this pilot study we use immunohistochemistry to measure the levels of activated FLT3 in bone marrow biopsies of patients prior to and during treatment inclinical trials at our institution to determine the efficacy of these FLT3 inhibitors and correlate it with patient outcomes. Results The different FLT3 compounds tested had a heterogenous effect on activated FLT3 levels. In some patients, ASP2215 caused a decrease in levels of activated FLT3 (indicated by brown nuclear staining), which corresponded to a decrease in leukemic blasts (Fig 1). Other patients, however, showed similar amounts of activated FLT3 both before and after treatment, which corresponded with no significant change in leukemic blasts. (Fig 2). Other FLT3 inhibitors also showed differences in their effects. Midostaurin reduced activated FLT3 levels, which correlated with a positive clinical response. In contrast, one patient receiving quizartinibshowed little to no decrease in activated FLT3 levels, despite remaining in clinical remission. This suggests that this FLT3 inhibitor may have alternative targets, such as the tyrosine kinases AXL or LTK. Conclusions The heterogeneity in the responses to ASP2215, midostaurin, and quizartinib suggests that there may be other targets for these compounds that are not currently accounted for in the clinical studies. Immunohistochemicalmeasurements of activated FLT3 in bone marrow sections before and after treatment with FLT3 inhibitors was not predictive for clinical response. Figure 1. Activated FLT3 levels in patient responsive to ASP2215; pre-treatment (A) and post-treatment (B) Figure 1. Activated FLT3 levels in patient responsive to ASP2215; pre-treatment (A) and post-treatment (B) Figure 2. Activated FLT3 levels in patient not responsive to ASP2215; pre-treatment (A) and post-treatment (B) Figure 2. Activated FLT3 levels in patient not responsive to ASP2215; pre-treatment (A) and post-treatment (B) Disclosures Larson: Pfizer: Consultancy; Ariad: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy.

scholarly journals Evaluation of a Bifunctional Sirpα-CD123 Fusion Antibody for the Elimination of Acute Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2544-2544 ◽  
Author(s):  
Siret Tahk ◽  
Saskia Schmitt ◽  
Christian Peter Augsberger ◽  
Binje Vick ◽  
Laia Pascual Ponce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Target Antigen ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.

scholarly journals A Standard, 3-Tube, 10-Color Flow Cytometry Panel for Acute Myeloid Leukemia Diagnosis Can be Used for MRD Detection at Day 30 after Induction, and Is Predictive of Early Relapse

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5336-5336
Author(s):  
Naheed Alam ◽  
Anne Tierens ◽  
Karen Yee ◽  
Aaron D. Schimmer ◽  
Vikas Gupta ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Induction Chemotherapy ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Early Relapse ◽  
Remission Status ◽  
One Year ◽  
Good Risk ◽  
Acute Myeloid

Abstract Introduction: As acute myeloid leukemia (AML) is a highly heterogeneous disease, strategies are needed to follow patients after induction and to predict relapse, so that second line treatments can be initiated early. Since assessment of remission status by morphology has known limitations, minimal residual disease (MRD) detection by flow cytometry (FC) has been used to evaluate remission status. In most publications, individually-tailored, patient-specific panels were applied at follow up. We evaluated MRD using a standard, 3-tube, 10-color antibody panel, which was applied at diagnosis and at day 30 post induction as the routine practice at Princess Margaret Cancer Center (PMCC). Methods: Patients who received front line induction chemotherapy for AML at PMCC from November 2012 to June 2013 and who were alive at Day 30 and able to undergo bone marrow aspiration were evaluated. All patients have had at least one year of follow-up. The leukemia associated immunophenotype was determined at diagnosis by a standard panel [AML1: CD65 FITC, CD13 PE, CD14 ECD, CD33 PC5.5, CD34 PC7, CD117 APC, CD7 A700, CD11b A750, CD16 PB, AML2: CD36 FITC, CD64 PE, CD56 ECD, CD33 PC5.5, CD34 PC7, CD123 APC, CD19 A700, CD38 A750, HLA-DR PB, CD45 KO, AML3: CD71 FITC, CD11c PE, CD4 ECD, CD33 PC5.5, CD34 PC7, CD2 APC, CD10 A700, CD235a A750, CD15 PB, CD45 KO with NaviosTM flow cytometer and KaluzaTM analysis software (Beckman Coulter)]. The analysis protocol was created individually for each patient to follow-up aberrant phenotypes detected at diagnosis. At follow up, 250x103 events were acquired, allowing a sensitivity level of 0.05%. A level of <0.1% cells with the leukemia-associated phenotype was used as the cutoff for MRD positivity, following previously published data. Results: The characteristics of 51 patients who were followed are summarized in Table 1. The median age of patients was 52 (range 18-81) years. Overall survival of all patients at one year was 70%. The cumulative incidence of relapse at one year was 22% (95% CI 8.0-34.1). Overall, 30 (59%) patients were positive for MRD by FC at day 30 after induction chemotherapy. Of these, 14 (47%) relapsed and 1 (3%) died in remission. Of 21 (41%) patients who were negative for MRD at day 30, only 4 (19%) relapsed, giving an odds ratio 3.72 [(95%CI 1.009-13.702), p-value 0.04]. Fifteen patients positive for MRD at day 30 remained in remission after 1 year. Of these, five (33%) were good risk patients with either inv(16) or t(8;21) abnormalities. Seven patients (47%) underwent allogeneic hematopoietic cell transplantation and remain in remission. The remaining three patients (20%) remain in remission without further intervention. Conclusion: MRD monitoring using standard, 3-tube, 10-color FC at day 30 after induction chemotherapy in AML is predictive of early relapse in standard and poor cytogenetic risk groups. In good risk cytogenetics patients, however, MRD positivity does not appear to be predictive. Table 1: Patient Characteristics n=51 Age at diagnosis, median (range), years 52 (18-81) AML classification (WHO 2008) Acute myeloid leukemia with inv(16)(p13.1q22) or t(16;16)(p13.1;q22), CBFB/MYH11 4 (8%) Acute myeloid leukemia with t(9;11)(p22;q23);MLLT3-MLL 1 (2%) Acute myeloid leukemia, t(8;21)(q22;q22) RUNX1-RUNX1T1 5 (10%) Acute myeloid leukemia with myelodysplasia-related changes 8 (15%) Acute myeloid leukemia not otherwise specified 12 (23%) Acute myeloid leukemia, NPM1 mutated, FLT3 ITD 9 (18%) Acute myeloid leukemia, NPM1 mutated, FLT3 TKD 2 (4%) Acute myeloid leukemia, NPM1 mutated, FLT3 negative 10 (20%) Cytogenetic Risk Good 5 (10%) Standard 39 (76%) Poor 7 (14%) WBC count at diagnosis, median (range) 16.7 (0.4-237) Hemoglobin at diagnosis, median (range) 88 (50-131) Platelet count at diagnosis, median (range) 36 (5-340) Neutrophil count at diagnosis, median (range) 1.4 (0-34.9) Peripheral blasts at diagnosis, median (range) 4.72 (0-197) Bone marrow blasts at diagnosis, median (range) 51.5 (20-95) Performance status 0-1 51 (100%) MRD + ve 30 (59%) MRD - ve 21(41%) Allogeneic HCT 16 (31%) Disclosures Yee: Roche: Research Funding. Gupta:Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Minden:Celgene: Honoraria. Porwit:Beckman-Coulter: Speakers Bureau.

scholarly journals Targeting and Depletion of Acute Myeloid Leukemia Blasts By MEN1112, a Novel Humanized Defucosylated Monoclonal Antibodies with Specificity for Bst1/CD157 Antigen

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2235-2235
Author(s):  
Adriano Venditti ◽  
Francesco Buccisano ◽  
Luca Maurillo ◽  
Maria Ilaria Del Principe ◽  
Andrea Coppola ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Distinct Pattern ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract MEN1112 is a new humanized, defucosylated, monoclonal antibody (mAb) with high specific affinity for Bst1/CD157 antigen. Bst1/CD157 antigen expression on blood cells of acute myeloid leukemia (AML) patients and healthy donors was investigated by flow cytometry using a PE-labeled MEN1112 mAb. Twenty three patients affected with AML have been tested, 18 at diagnosis, 4 at relapse, 1 resistant. In 16 out of 23 patients both bone marrow (BM) and peripheral blood (PB) specimens were evaluated. PB and BM samples from healthy donors (N=2) were also assessed. In healthy donors and AML patients, PB and BM lymphocytes were Bst1/CD157 antigen negative whereas monocytes and neutrophils showed a distinct pattern of MEN1112 mean fluorescence intensity (MIF), with monocytes having the brightest expression. In the stem cell compartment, an intermediate level of MFI was observed (p<0.001). Prevalence of expression of the antigen on patients’ samples was over 90%. On AML blast cells from each single patient, MEN1112 expression was heterogeneous; indeed the antigen was expressed on 50%±29% and 47% ±39% of blasts in BM and PB, respectively. The anti-leukemia activity of MEN1112 on AML cell lines was tested, in vitro, by a flow cytometry-based cell depletion assay in the presence of lymphokine activated immune effector cells: a strong depletion of leukemia cells was demonstrated suggesting that MEN1112 might exert anti-leukemia activity through antibody dependent cell-mediated cytotoxicty (ADCC). The activity of MEN1112 was also tested ex vivo on whole PB showing that the antibody was able to deplete AML blasts in 9 out of 23 patients (47.4 %) with a percentage of AML blast depletion ranging between 4.3 - 66 %. In whole BM from 2 out of 11 evaluable patients MEN1112 induced 68% and 23% of AML blast depletion. Bst1/CD157 shedding assessment showed that, in the sera from AML samples, the concentration of Bst1/CD157 antigen was comparable to that measured in healthy donors. Moreover, since Fcγ receptor (CD16) genotype might be a factor contributing to the antitumor activity of the antibody, the polymorphism CD16-158Phe/Val was analyzed. Five out of 19 samples were homozygous for CD16-158 Phe; 5 were homozygous for CD16- 158 Val and 9 were heterozygous for CD16-158. MEN1112-induced blast depletion was observed for each genotype. Moreover, in an attempt to identify the determinant of MEN1112 activity, % in PB of blast (antigen positive), NK cells or residual normal cells were evaluated. Altogether, these results are promising suggesting the potential for an ADCC-mediated MEN1112 antileukemic effect and they support the clinical development of MEN1112. Disclosures Venditti: Menarini Ricerche SpA: Research Funding. Buccisano:Menarini Ricerche SpA: Research Funding. Del Principe:Menarini Ricerche SpA: Research Funding. Coppola:Menarini Ricerche SpA: Research Funding. Palomba:Menarini Ricerche SpA: Research Funding. Aureli:Menarini Ricerche SpA: Research Funding. Arriga:Menarini Ricerche SpA: Research Funding. Bellarosa:Menarini Spa: Employment. Bressan:Menarini Ricerche SpA: Employment. Manzini:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche SpA: Employment. Amadori:Menarini Ricerche SpA: Research Funding. Sconocchia:Menarini Ricerche SpA: Research Funding.

Ara-C Treatment of Acute Myeloid Leukemia Does Not Lead to Prolonged Enrichment of Stem Cells or a Cell Cycle Arrest

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2178-2178
Author(s):  
Robin L. Perry ◽  
Jean-Emmanuel Sarry ◽  
Gwenn-ael Danet-Desnoyers ◽  
Martin Carroll
Keyword(s):  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Single Agent ◽  
Chemotherapy Resistance ◽  
Post Treatment ◽  
Chemotherapy Treatment ◽  
New Therapies ◽  
Acute Myeloid

Abstract Abstract 2178 The major therapeutic barrier in acute myeloid leukemia is chemotherapy resistance. Although most patients will respond to treatment with chemotherapy over 50% of responders will relapse and eventually die of disease. Many hypotheses have been proposed to explain chemotherapy resistance but none of these have lead to new therapies or a complete understanding of the molecular mechanisms of AML chemotherapy resistance. In order to develop an improved understanding of chemotherapy resistance in AML, we have developed a chemotherapy model of human AML in NSG mice. Mice are engrafted with primary AML samples from patients seen at diagnosis or relapse of disease or who demonstrated primary chemotherapy resistance. After demonstrating AML engraftment, mice are treated with cytosine arabinoside (Ara-C) given IP daily for 5 days as a single agent at 10mg/kg daily, 30mg/kg daily, and 60 mg/kg daily which correlates with human dosing. In >75% of mice treated with 10 mg/kg of Ara-C there is a cytoreductive effect at 2 weeks post-treatment with relapse at 4 weeks post-treatment. In all mice treated with 30 mg/kg of Ara-C there was a cytoreductive effect at 2 weeks post-treatment with relapse at 4 weeks post-treatment. In all mice treated with 60 mg/kg of Ara-C there was a cytoreductive effect at 2 weeks post-treatment with relapse 4–13 weeks post-treatment when relapse occurred, demonstrating that there is a dose response relationship in the model in terms of nadir leukemic burden and time to peripheral blood relapse. Two weeks after treatment, there was up to a 50-fold decrease in total AML cell burden in the peripheral blood of mice treated with 30 mg/kg and and up to a 70-fold decrease in the peripheral blood of mice treated with 60 mg/kg of Ara-C. We found no enhancement in quiescent or G0 cells after chemotherapy treatment. We did however, in 1/5 samples tested see a change in phenotype after chemotherapy treatment with an increase in the total number of CD34+38+ cells with a concomitant decrease in CD34-38+ cells. In all other samples tested, there was no change in phenotype after chemotherapy treatment. These results are in contrast to recent studies using a 1 gm/kg dose of Ara-C and analysis at Day 3 following a single treatment. These results have implications for understanding the physiologic response to Ara-C at different doses. We are currently analyzing mRNA expression arrays of AML without or with Ara-C treatment to identify novel mechanisms of chemotherapy resistance. Taken together, this model provides a novel approach for development of new therapies in AML. Disclosures: Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

scholarly journals Associating Ex Vivo Drug Sensitivity with Differentiation Status Identifies Effective Drug Combinations for Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1301-1301
Author(s):  
Christopher A. Eide ◽  
Stephen E. Kurtz ◽  
Andy Kaempf ◽  
Nicola Long ◽  
Daniel Bottomly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Single Agent ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p&lt;0.0001). This broad sensitivity of the VEN+DORA combination was not significantly associated with an array of clinical, genetic, and mutational features in the patient samples tested, in contrast to single agents, particularly VEN. Analysis of blood cell differential counts of patient samples tested identified increased monocyte levels were significantly correlated with sensitivity to DORA and resistance to VEN as single agents (Spearman r = -0.3 and 0.6; p&lt;0.0001), associations that were not apparent with the combination. For patient samples with accompanying FAB differentiation state-based designations (n=108), sensitivities of the combination were similar across classifications of undifferentiated (M0/M1) through monocytic (M4/M5) acute leukemia. In contrast, single-agent VEN was significantly more sensitive in undifferentiated compared to monocytic specimens, whereas DORA sensitivity showed the reverse trend (though to a lesser degree). These differences in sensitivity were further validated by immunophenotyping data where available (n=105), which showed surface markers associated with resistance to VEN (CD11b, CD14, CD16, CD56, CD64, HLADR; Wilcoxon Rank Sum, p&lt;0.001 to p=0.007) or sensitivity to VEN (CD117; p=0.001) or DORA (CD14; HLADR; p=0.004). By contrast, none of these associations significantly distinguished sensitivity for the VEN+DORA combination. Expression levels of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were concordant with their respective drug sensitivities associated with FAB classification; that is, significantly higher levels of BCL2 in M0/M1 leukemias and MAPK14 in M4/M5 cases (Mann-Whitney test; p&lt;0.0001; n=145). Further dissection of transcriptomic and drug sensitivity data revealed strong correlation and gene set enrichment for DORA and VEN sensitivities with monocyte-like and progenitor-like signatures, respectively (n=225), for cell differentiation states previously described for AML (van Galen 2019), and these associations diminished for the combination treatment. Lastly, the VEN+DORA combination enhanced efficacy and synergistic inhibition was confirmed using human AML cell line models tested with a matrix of potential dose concentrations. Taken together, these findings suggest that exploiting distinct, complementary sensitivity profiles of targeted therapies with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers an opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML. Disclosures Druker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

scholarly journals CMV reactivation after allogeneic HCT and relapse risk: evidence for early protection in acute myeloid leukemia

Blood ◽  
2013 ◽  
Vol 122 (7) ◽  
pp. 1316-1324 ◽  
Author(s):  
Margaret L. Green ◽  
Wendy M. Leisenring ◽  
Hu Xie ◽  
Roland B. Walter ◽  
Marco Mielcarek ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Early Relapse ◽  
Relapse Risk ◽  
Allogeneic Hct ◽  
Increased Risk ◽  
Key Points ◽  
Cmv Reactivation ◽  
Reduced Risk ◽  
Acute Myeloid

Key Points CMV reactivation after HCT is associated with a reduced risk of early relapse in patients with AML but not other disease groups. The benefit, however, is offset by an increased risk of nonrelapse mortality.

scholarly journals Discovery of a Novel Dihydroorotate Dehydrogenase Inhibitor That Induces Differentiation and Overcomes Ara-C Resistance of Acute Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2663-2663
Author(s):  
Satoshi Kitazawa ◽  
Yukiko Ishii ◽  
Keiko Makita-Suzuki ◽  
Koichi Saito ◽  
Kensuke Takayanagi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Oral Administration ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Dihydroorotate Dehydrogenase ◽  
Once Daily ◽  
Hl60 Cells ◽  
Pyrimidine Nucleotide ◽  
Acute Myeloid

Cancer initiating cells (CIC) are suggested to be responsible for drug resistance and cancer relapse that are associated with poor prognosis. Therefore, drugs effective for CIC could fulfill an unmet clinical need. We performed a drug screen with chemical libraries to find out new compounds which specifically eradicated CIC established in the previous report (Yamashita et al., Cancer Research, 2015). We obtained compounds with a carboxylic acid skeleton as hit compounds. Interestingly, FF1215T, one of the hit compounds, was shown to inhibit growths of CIC by decreasing intracellular pyrimidine nucleotide levels. Finally, we identified dihydroorotate dehydrogenase (DHODH), which was essential for de novo pyrimidine synthesis as the target of the hit compounds in a ligand fishing assay. FF1215T inhibited DHODH enzymatic activity with the 50% inhibitory concentration value of 9 nM, which showed greater potency than well-known DHODH inhibitors brequinar (12 nM), teriflunomide (262 nM), and vidofludimus (141 nM). Growing evidence suggests that DHODH is considered to be a promising target to overcome a differentiation blockade of acute myeloid leukemia (AML) cells (Sykes et al., Cell, 2016).Therefore, we explored the effect of FF1215T on AML growth and differentiation. FF1215T demonstrated growth inhibitory effect in multiple human AML cell lines such as U937, MOLM13, HL60, and MV4-11 with the 50% growth inhibition values of 90-170 nM. FF1215T decreased intracellular pyrimidine nucleotide levels, induced DNA damage marker γ-H2AX possibly due to the replication stress, and finally led to apoptosis in HL60 cells. Cell cycle analysis revealed that FF1215T treatment arrested HL60 and THP1 cells at S phase and increased sub-G1 population in these cells. In addition, our DHODH inhibitors induced upregulation of cell-surface CD11b and CD86, which are monocyte and macrophage differentiation markers, morphological changes, and phagocytic activities in several AML cells, indicating differentiation of AML cells toward monocyte and macrophage by DHODH inhibition. FF1215T also depleted UDP-GlcNAc, a substrate for Protein O-GlcNAcylation, and diminished global O-GlcNAcylation and O-GlcNAcylated protein expressions such as c-Myc, SOX2, and OCT4, which play important roles in maintenance and self renewal of stem cells. We also found that our DHODH inhibitors induced CD11b and CD86, and increased the ratio of macrophage-like cells in primary patient-derived AML cells and these effects were rescued by uridine supplementation (Fig). Inhibitions of colony formations of primary AML cells were also shown after 14 days of FF1215T treatment. In exploring the value of DHODH inhibitors in the clinic, we identified that our DHODH inhibitors worked to overcome the resistance of standard therapy Ara-C. Our DHODH inhibitors were effective against Ara-C-resistant models of HL60 cells as well as HL60 parental cells. Notably, our DHODH inhibitors synergistically inhibited growths of Ara-C-resistant THP1 cells and enhanced CD11b upregulation of THP1 cells when combined with Ara-C by activating conversion of Ara-C to its active form Ara-CTP. Next, we optimized the hit compounds and identified an orally available DHODH inhibitor FF14984T that achieved high and prolonged plasma concentrations in vivo. Oral administration of 10 and 30 mg/kg FF14984T once daily for 10 days exhibited significant anti-tumor effects in mice xenografted with HL60 cells. These treatments showed strong reduction of CTP in tumor and induction of DHO in tumor and plasma. When 30 mg/kg FF14984T was orally administrated to orthotropic MOLM13-xenografted mice once daily for 12 days, hCD45+ cells proportions in bone marrow were decreased whereas hCD11bhigh/hCD45+ ratio increased, indicating that FF14984T induced AML differentiation in vivo. Finally, oral administration of 30 mg/kg FF14984T once daily significantly prolonged survival of mice in U937 orthotropic models. Taken together, we developed a novel potent DHODH inhibitor FF14984T that induced cellular differentiation and anti-leukemic effects on cell lines and primary AML cells. FF14984T is possibly a promising therapeutic option for Ara-C-resistant AML patients that can also benefit from the combination therapy of FF14984T and Ara-C. Identifying the precise mechanism of AML differentiation by DHODH inhibitor and its effects on CIC are currently ongoing. Disclosures Kitazawa: FUJIFILM Corporation: Employment. Ishii:FUJIFILM Corporation: Employment. Makita-Suzuki:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Takayanagi:FUJIFILM Corporation: Employment. Sugihara:FUJIFILM Corporation: Employment. Matsuda:FUJIFILM Corporation: Employment. Yamakawa:FUJIFILM Corporation: Employment. Tsutsui:FUJIFILM Corporation: Employment. Tanaka:FUJIFILM Corporation: Employment. Hatta:FUJIFILM Corporation: Research Funding. Natsume:FUJIFILM Corporation: Research Funding. Kondo:FUJIFILM Corporation: Research Funding. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Perseus Proteomics Inc.: Research Funding.

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