Normal Marrow Progenitor Research Articles

A phase 1b/2 study of pivekimab sunirine (PVEK, IMGN632) in combination with venetoclax/azacitidine for patients with newly diagnosed CD123-positive acute myeloid leukemia.

TPS6585 Background: Despite improved outcomes with azacitidine (AZA) and venetoclax (VEN) in newly diagnosed (ND) unfit acute myeloid leukemia (AML), only a subset of patients (pts) respond (CR 37%; CR/CRi 66%) and long-term survival remains inadequate (mOS <15m, DiNardo NEJM 2020). The measurable residual disease (MRD)-negative rate was 41% in AZA-VEN treated pts in VIALE-A which was associated with improved survival (Pratz JCO 2022). CD123 is expressed on the majority of AML blasts and leukemic stem cells while minimally expressed on normal hematopoietic stem cells (Kovtun Blood Adv 2018). Pivekimab sunirine (PVEK, IMGN632) is an antibody-drug conjugate comprising a high-affinity CD123 antibody, cleavable linker, and an indolinobenzodiazepine pseudodimer (IGN) payload. The IGN payload alkylates DNA and causes single strand breaks without crosslinking. IGNs are designed to have high potency against tumor cells, while demonstrating less toxicity to normal marrow progenitors than other DNA-targeting payloads. Clinical data from the first 50 pts in the dose expansion cohorts 1 and 2 with ND AML demonstrated a 76% (22/29) MRD negativity rate (by flow cytometry, negativity threshold <0.1%) (Daver ASH 2023), supporting the continued enrollment of the PVEK+AZA+VEN triplet and further evaluation of the regimen’s antileukemia activity and safety in consideration of potential registration-enabling trials. Methods: This is an open-label, multicenter, phase 1b/2 study of PVEK administered in a combination with AZA+VEN in pts with ND CD123-positive (CD123+ by flow cytometry or IHC) AML, with no prior treatment with hypomethylating agents (HMA). Pts will receive the recommended phase 2 dose of PVEK 0.045 mg/kg IV as a < 30-minute outpatient infusion on day 7, AZA 75 mg/m2 SC or IV daily on days 1 to 7, and VEN 400 mg PO daily for up to 28 days in a 28-day cycle. During cycle 1, a bone marrow evaluation at or around day 21 is required to inform VEN dose duration. Current eligibility criteria for continued enrollment of AML patients unfit for intensive chemotherapy include age ≥ 75 years old, or age < 75 years old with ECOG 2-3, or at least one defined comorbidity. The primary study objectives are to assess antileukemia clinical activity (composite CR [complete remission] rate, overall response rate, duration of remission) and MRD-negativity rates. Key secondary objectives are safety and tolerability, pharmacokinetics and immunogenicity. The PVEK+AZA+VEN triplet in pts with ND unfit AML is currently enrolling across sites in France, Germany, Italy, Spain, UK and USA. Clinical trial information: NCT04086264 .

A phase 1b/2 study of pivekimab sunirine (PVEK, IMGN632) in combination with venetoclax/azacitidine or magrolimab for patients with CD123-positive acute myeloid leukemia (AML).

TPS7073 Background: Despite improved outcomes with azacitidine (AZA) and venetoclax (VEN) in frontline unfit AML (CR 37% and CR/CRi 66%), long-term survival for these patients remains inadequate (mOS < 15m, DiNardo NEJM 2020). CD123 is expressed on the majority of AML blasts and leukemic stem cells while minimally expressed on normal hematopoietic stem cells (Kovtun Blood Adv 2018). Pivekimab sunirine (PVEK, IMGN632) is an antibody-drug conjugate (ADC) comprising a high-affinity CD123 antibody, cleavable linker, and an indolinobenzodiazepine pseudodimer (IGN) payload. The IGN payload alkylates DNA and causes single strand breaks without crosslinking. IGNs are designed to have high potency against tumor cells, while demonstrating less toxicity to normal marrow progenitors than other DNA-targeting payloads. Clinical data in relapsed/refractory (R/R) AML (Daver ASH 2022) supported the continued clinical exploration of the PVEK+AZA+VEN triplet. The current enrolling dose expansion cohorts (Regimen C; Cohorts 1 and 2) in frontline AML will evaluate antileukemia activity and MRD levels in the combination of PVEK+AZA+VEN. Regimen E will enroll patients with R/R AML to evaluate the safety and antileukemia activity of PVEK in combination with magrolimab. Methods: This is an open-label, multicenter, Phase 1b/2 study of PVEK administered in combination regimens in patients with frontline and R/R CD123-positive AML. Patients will receive the established recommended phase 2 dose (RP2D) of PVEK 0.045 mg/kg IV, as a < 30-minute outpatient infusion. In Regimen C, patients will receive PVEK 0.045 mg/kg IV on D7 + azacitidine 75 mg/m2 SC or IV daily on days 1 to 7, and venetoclax up to 400 mg PO daily for at least 14 days (Cohort 1) or for up to 28 days (Cohort 2) in a 28-day cycle. In Regimen E, R/R AML patients will receive PVEK 0.045 mg/kg IV on D1 of the 28-day cycle in combination with magrolimab at 30 mg/kg Q2W, after the standard ramp up schedule. Regimen C will enroll adults with untreated AML, CD123+ by flow cytometry or IHC and no prior HMA treatments. The primary endpoints for Regimen C are composite CR rate, overall response rate (ORR), duration of remission (DOR), MRD levels and key secondary endpoints are safety, PK and immunogenicity. Regimen E will enroll adult patients with relapsed or refractory CD123+ AML with up to 2 prior lines of therapy. The primary endpoint for Regimen E is dose limiting toxicities (DLTs) and key secondary endpoints are safety, antileukemic activity, PK and immunogenicity. The PVEK+AZA+VEN triplet (Regimen C) is currently enrolling frontline unfit patients across sites in France, Germany, Italy, Spain, UK and USA. The PVEK+Magro doublet (Regimen E) is planned to be open for enrollment mid-2023 at sites in the USA. Clinical trial information: NCT04086264 .

Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating Factor Is Toxic to Blasts From Patients With Juvenile Myelomonocytic Leukemia and Chronic Myelomonocytic Leukemia

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10-9 mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units–leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit–granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34+ cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34+ bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor α and/or β chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF–induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.

PPM1D Truncating Mutations Confer Chemotherapy Resistance in Hematopoietic Stem Cells, Which Is Reversible By PPM1D Inhibition

One of the adverse consequences of chemotherapy exposure is the development of therapy-related myeloid neoplasms (t-MNs). However, the cause and origin of most t-MNs are unknown, and the prognosis remains dismal. Novel work has shown that in addition to TP53, PPM1D is selectively mutated in 15% of therapy related MDS (Lindsley et al., ASH Abstract). Truncating mutations of PPM1D are also found to commonly occur in clonal hematopoiesis (Jaiswal et al., NEJM 2014; Genovese et al., NEJM 2014; Xie et al., Nat Med 2014), as well as in the blood of cancer patients, particularly after exposure to chemotherapy (Ruark et al., Nature 2013; Swisher et al., JAMA Oncol 2016). We hypothesized that PPM1D truncating mutations confer chemotherapy resistance, causing selective outgrowth of PPM1D mutant hematopoietic stem cells in states of genotoxic stress. In addition, since PPM1D mutations lead to a gain of function, we examined the potential of targeting PPM1D-mutant cells pharmacologically.The protein phosphatase PPM1Dis a direct regulator of TP53 activity and the DNA damage response pathway (Fiscella et al., PNAS 1997). Consequently, gain-of-function PPM1D mutations lead to decreased TP53 activity. To examine whether PPM1D mutations drive chemotherapy resistance through an abrogation of the TP53 dependent DNA damage response, we engineered PPM1D-mutant subclones using the CRISPR-Cas9 system in the TP53 wild-type AML cell line MOLM-13. PPM1D exon 6 truncation led to increased expression of PPM1D and resistance to DNA damaging agents, including cytarabine, cyclophosphamide and cisplatin. In addition, PPM1D-mutant cells exhibited a selective advantage over wild-type cells in the presence of chemotherapy, expanding 100-fold over a 24-day period (Fig. 1). While treatment with chemotherapy induced phosphorylation of Chk1 and p53, cell cycle arrest and apoptosis in wild-type cells, this response was abrogated in PPM1D-mutant cells due to gain of function of PPM1D. Using phosphoproteomic analysis, we further demonstrate decreased phosphorylation of known and novel targets in PPM1D-mutant compared to wild-type cells.We next investigated the effect of PPM1D mutation on normal marrow progenitors in response to chemotherapy treatment in vivo. We performed a competition experiment in which mouse bone marrow c-Kit+ cells expressing Cas9 were transduced with guide RNAs targeting exon 6 of PPM1D or a control guide, and then transplanted into mice in a 1:5 ratio. We observed a selective outgrowth of PPM1D-mutant myeloid cells in the peripheral blood of mice after exposure to chemotherapy. In addition, we found expansion of PPM1D-mutant cells in the lineage negative, c-Kit+ Sca-1+ population, which is enriched for hematopoietic stem cells and multipotent progenitors, indicating that PPM1D-mutant stem and progenitor cells have a competitive advantage over wild-type cells after exposure to genotoxic stress.The generation of a selective, allosteric inhibitor of PPM1D (Gilmartin et al., Nat Chem Biol 2014) allowed us to examine whether pharmacologic inhibition of PPM1D decreases the chemotherapy resistance or survival of PPM1D-mutant cells. We found that PPM1D-mutant cells have a significantly increased sensitivity to PPM1D inhibition when compared to wild-type controls. In addition, PPM1D inhibitor treatment was able to re-sensitize mutant cells to chemotherapy and abrogate the selective outgrowth of PPM1D-mutant cells during chemotherapy exposure. Lastly, we demonstrate that the proteome-wide phosphorylation profile characteristic of PPM1D-mutant cells can be reversed through treatment with the PPM1D inhibitor.In sum, these results demonstrate that PPM1D mutations confer a competitive advantage to hematopoietic stem cells undergoing genotoxic stress by abrogating the DNA damage response, and are likely to be the initiating mutation in a large proportion of t-MNs. Due to the gain-of-function nature of this mutation, PPM1D-mutant cells are differentially sensitive to treatment with a PPM1D inhibitor. PPM1D inhibition may therefore provide an opportunity for the prevention and targeted treatment of hematologic malignancies that harbor PPM1D mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combined inhibition of the phosphoinosityl-3-kinase (PI3Kinase) P110δ subunit and mitogen-extracellular activated protein kinase (MEKinase) shows synergistic cytotoxicity against human acute myeloid leukemia progenitors

Treatment of 32 acute myeloid leukemia (AML) blast samples showing activation of the PI3K and RAS/RAF/MEK/ERK pathways with the PI3K p110δ isoform and MEKinase selective inhibitors, PCN5603 and U0126 produced dose dependent progenitor kill and inhibition of p-Akt Ser473 and p-Erk Tyr204 expression. Normal marrow or blood progenitors were less sensitive to these inhibitors (median PCN5603 IC50s for AML and normal cells 1.5 and 5.8μM and for U0126 9.6 and 25.8μM, respectively). U0126 synergized with PCN5603 for killing of both AML and normal progenitors. However, the synergy was less for normal than for AML cells and the median IC50 of each drug in the combination 7- to 10-fold higher than for AML cells.

Evaluation of Rigosertib in Patients with a Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML) Relapsed or Refractory to Hypomethylating Agents: A Phase I/II Study

Abstract 3794 Background:Rigosertib is a small molecule anti-cancer agent with a multi-targeted mechanism of action. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to rigosertib compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Chen Proc AACR 2008). Azacitidine is first-line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating agents have a short life expectancy of approximately 4 to 6 months (Jabbour 2010, Prebet 2011). There are no approved second line therapies for this patient population. Methods:A phase I/II study of Rigosertib is being conducted in pts with MDS and AML. Pts with higher-risk disease had to have failed a hypomethylating agent. In the phase I component, pts were entered in cohorts of escalating doses in a classic 3+3 design ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments were administered every 3 to 4 weeks. A maximum tolerated dose of 1375 mg/m2 was identified for the phase II component, and subsequent pts were treated with this dose as a CIV for 72 hours. A CBC was performed weekly and a bone marrow (BM) was performed at baseline and week 4, 8, and then q3 months afterwards. Results:Twenty-one patients with MDS or AML refractory/relapsed to a hypomethylating agent have been treated with rigosertib. The study cohort comprised pts with a diagnosis of intermediate-2 MDS (2 pts), high risk MDS (5 pts), chronic myelomonocytic leukemia (1 pt), and AML (13 pts) (all AML had an antecedent MDS). The median age was 79 years. 86% of pts were male. Patients received between 1–19 cycles of treatment. Their cytogenetic profiles were diverse with the most recurrent abnormalities including a complex karyotype (5 pts), normal (5 pts), monosomy 7 (4 pts), and trisomy 8 (2 pts). Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: marrow CR (4), hematologic improvement (HI) (2); erythroid (1) platelet (1). Time to response was 2–4 cycles. An additional 2 pts had a >50% BM blast decrease from baseline but not to < 5%. Three pts had stable disease after treatment but their courses were complicated by infections requiring hospitalization and removal from the study. Three pts were deemed to be inevaluable because they received less than 2 cycles of treatment or did not have a follow-up bone marrow evaluation. Thus, 9/18 evaluable pts (50%) demonstrated either a bone marrow/peripheral blood response (6) or stable disease (3). The median overall survival of those with marrow CR+PR was 10.1 months versus 2 months for those without a bone marrow response (p=0.0011, log-rank test). Of those pts who did not respond or were inevaluable, the majority (83%) had AML, many with a proliferative course. The most frequent grade 1–2 side-effects included dysuria, hematuria, fatigue, anorexia, nausea, and diarrhea. Possibly related grade 3 side-effects included fatigue, hematuria, and dyspnea, each in one pt. Six of 21 pts developed cystitis manifested by dysuria and/or hematuria. Among responding or stable patients, 5 of 9 had cystitis compared with 1 of 12 non-responders. Patients who developed symptomatic cystitis were treated with sodium bicarbonate with improvement. The relationship between dysuria and/or cystitis and response is being investigated. Conclusions:Rigosertib appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. It has biologic activity with reduction in BM blasts and improvement in the peripheral blood counts in a subset of treated pts, and these effects are associated with increased survival. Dysuria/cystitis may be a response related biomarker and requires further analysis. Data regarding pharmacokinetics and pharmacodynamics will be presented and correlated to response. Given promising initial results, a phase III multicenter randomized trial is underway to compare rigosertib to best supportive care with a primary endpoint of overall survival in patients with higher risk MDS who have failed, progressed, or relapsed after treatment with hypomethylating agents. Disclosures:Reddy:Onconova: Research Funding. Holland:Onconova: Research Funding. Wilhelm:Onconova: Employment, Equity Ownership. Silverman:Onconova: Research Funding.

Abstract 1350: Transgenic expression of PKR induces a myelodysplastic syndrome/pre-leukemia-like condition in mice

Abstract Double stranded (ds) RNA activated protein kinase PKR was discovered over 30 years ago as a downstream mediator of the interferon signal pathway. Initial studies focused on PKR's anti-virus and pro-inflammatory effects. More recently, PKR has been found to be involved in signal transduction pathways critical for the negative regulation of cell growth and initiation of apoptosis. Significantly, loss of PKR expression/activity has been associated with increased growth of human breast carcinoma, B-cell CLL, and T-cell ALL using patient samples. Furthermore, PKR activity is increased in bone marrow progenitor cells isolated from patients with myelodysplastic syndrome (MDS) which undergo significant apoptosis compared to normal marrow progenitors not observed when evolved to AML. Thus, in the process of MDS evolution to leukemia, PKR expression/activity may be downregulated by a process that is currently unclear. To explore the role of PKR in MDS/AML we have constructed a transgenic mouse model expressing human PKR (TgPKR) under control of vav regulatory elements to drive expression specifically in hematopoietic-tissue. We hypothesized transgenic expression of human PKR would exert an inhibitory effect on mouse hematopoiesis, cause ineffective blood cell production, and thus simulate a disease process similar to human MDS. In our preliminary data, quantitative PCR and immunoblotting results confirm that human PKR is specifically expressed in spleen, thymus and bone marrow (BM) but not in liver, kidney, intestine, muscle and other tissues outside hematopoiesis in TgPKR mice. Aberrant activation of transcription factor STAT1 and MAPKinases ERK1/2 occurs in hematopoietic cells from the TgPKR mice. In addition, inhibitory cytokines including interferon gamma, CXCL9 and 10 are overexpressed. Initial characterization of BM cells from TgPKR mice indicates a higher level of uninduced/basal apoptosis in growth medium and increased apoptosis upon withdrawal of growth factors compared to cells from wild type (WT) littermates. Furthermore, BM cells from TgPKR mice display &amp;gt;20% reduced colonies (CFU-GM and CFU-GEMM) which contain 40% less cells than those from WT littermates. Significantly, histopathological analysis of BM and spleen from TgPKR mice reveals a ∼20% increase in bone marrow cellularity with a striking increase in dysplastic megakaryocytes that are either small without nuclear lobation or of normal size but with hypolobation or separated nuclear lobes. Taken together our results demonstrate that transgenic expression of hPKR induces an MDS-like condition in mice characterized by reduced BM cell survival, increased apoptosis and reduced proliferation and differentiation of BM progenitor/stem cells. Thus, increased PKR expression in patients may, at least in part, lead to a BM failure state similar to MDS. Thus, future anti-MDS therapies that inhibit PKR may be clinically useful. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1350. doi:1538-7445.AM2012-1350

Aminopeptidase inhibition by the novel agent CHR-2797 (tosedostat) for the therapy of acute myeloid leukemia

Aminopeptidase enzyme inhibition is thought to deplete the free intracellular amino acids needed by malignant cells for growth and development, resulting in profound anti-proliferative and apoptotic effects. In this study, we investigated the effects of the metalloenzyme-inhibitor CHR-2797 (tosedostat), in primary acute myeloid leukemia (AML) cells. CHR-2797 demonstrated marked in vitro cytotoxicity in AML samples and strong synergy with Cytarabine (Ara-C), but significantly less cytotoxicity to normal marrow progenitors. Furthermore mechanistic investigations revealed that CHR-2797 inhibited the intrinsic nuclear, cytoplasmic and cell surface aminopeptidase function of AML blasts in a dose-dependent manner, demonstrating a promising novel approach for AML therapy.

Evaluation of ON01910.Na In Patients with a Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML) Relapsed or Refractory to Hypomethylating Agents: A Phase I Study

AbstractAbstract 2944 Background:ON 01910.Na a novel benzyl styryl sulfone derivative is under clinical development in hematologic malignancies. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to ON 01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). Azacitidine (AzaC), is first line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating based therapies have a poor prognosis and there are no accepted effective second line treatments, thus a need for new agents. Methods:A phase I/II study of ON 01910.Na is being conducted in pts with hematological malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. A CBC is performed weekly and a bone marrow (BM) is performed at baseline and week 4, 8, and then q3 months thereafter. Pts with higher-risk disease had to have failed a hypomethylating agent. Results:Ten pts with MDS or AML relapsed/refractory to a hypomethylating agent have been treated with ON 01910.Na thus far (table 1). The study cohort comprised pts with a diagnosis (Dx) RAEB-2 (4 pts), RAEB-T (1 pt), and AML (5 pts) (median age of 75 years). Their cytogenetic profile included 1 pt with normal, 2 with intermediate (+8), and 7 pts with poor risk cytogenetics (monosomy 7 and/or complex). Patients were treated between 5 and 70 weeks. Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: Marrow CR (3), hematologic improvement (HI-P) (2); erythroid (1) platelet (1). An additional 2 pts had a >50% BM blast decrease from baseline but not to <5%. Thus, 5/10 (50%) demonstrate a bone marrow response. Survival of these pts was 7.3, 15.7, and 16.4 months; one patient remains on study 5+ months. Four of the five responders had MDS at the initiation of treatment: RAEB-2 (3), CMMoL (1), AML (1). Responders had monosomy 7 (2), trisomy 8 (1) and complex cytogenetics (2). One pt had an elimination of the MDS clone and the others had persistence of the abnormal karyotype throughout their treatment course. Five pts had SD without HI at 4 weeks, 2 pts progressed to AML. All 5 non-responders had AML; 4 with a proliferative course. These latter received only 2 (2) or 3 (3) cycles before succumbing to disease related infectious complications. Survival for these patients ranged from 1.3 – 2 months with a median duration on study of 42 days. The most frequent side effects grade2 2 for all pts included fatigue, anorexia, nausea, and dysuria in patients receiving extended duration infusions. One pt had a grade 3 urinary frequency. No hematologic toxicities occurred and no bone marrow toxicity or hypoplasia was noted. Pharmacokinetic studies are ongoing, data to date demonstrate no evidence of drug accumulation in patients who are treated repeatedly. Conclusion:ON 01910.Na appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. ON 01910.Na has biologic activity with reduction in BM blasts, eradication of the MDS clone and improvement in the peripheral blood counts in some pts. These effects are associated with increased survival albeit in limited numbers of pts treated thus far. Further study of ON 01910.Na is warranted to better define biologic activity, appropriate target populations and to define mechanism of action.Table 1Pt IDInitial Dx/OnStudy DxPrior TherapyOn Study % BM BlastsMax BM Response (IWG 2006 criteria)Dosing Cohorts (mg/m2/d) CIV# of cyclesDuration On Study (mo)OS (mo)001High/AMLAzaC80PR6501077.3002Int-1 MF/highAzaC11CR10501415.715.7003AML/AMLAzaC + saha45NR105021.62004High/highAzaC17CR10501916.416.4005Int-2/AMLAzaC91NR137521.21.5006CMMoL/CMMoLDecitabine22PR137541.64.7007Int-2/AMLAzaC66NR137531.41.7008Int-1/AMLAzaC44NR170031.41.7009MDS-MF-AML/AMLDecitabine51NR170031.21.3010Int-2/highAzaC & decitabine15CR137575+5+ Disclosures:Silverman:Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.

Effects of a Novel Benzyl Styryl Sulfone Derivative ON 01910.Na On the Myelodysplastic Syndrome (MDS) Derived Clone in Patients Relapsing Following Response to Azacitidine (AzaC) Therapy.

Abstract 4839 BackgroundON 01910.Na, a novel benzyl styryl sulfone derivative is currently under clinical development as an anticancer agent in solid tumors and hematologic malignancies. ON 01910.Na selectively targets tumor cells at the mitotic phase of the cell cycle, arrests leukemia cells at the G2/M stage and induces apoptosis in a time and dose dependent manner. ON 01910.Na affects multiple cellular pathways, has multikinase activity and has been shown to target multiple genetic pathways, including those that are regulated by cyclin dependent kinases. Our studies indicate that leukemic cells exhibit significantly higher levels of sensitivity of ON01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). AzaC alters the natural history of higher risk MDS with effects on marrow function, reduction in the risk of leukemic transformation and increased overall survival (Silverman JCO 2002, 2006; Fenaux Lancet 2009). However, 50% of patients (pts) do not respond to AzaC and upon progression after treatment their prognosis is poor, thus, a need for alternative agents. MethodsA phase I/II study of ON 01910.Na is being conducted in pts with heme malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CI) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. ResultsThis report details two pts with progressive disease following treatment for MDS with AzaC at a dose of 75mg/m2/d x 7 days SC every 4 weeks. Upon progression they were treated with ON 01910.Na at 1050 mg/m2/d CI x 72h to 144 hours. Patient 002 presented with MDS int-2 with myelofibrosis and mild splenomegaly, 8% marrow blasts, normal karyotype, and RBC transfusion dependence. AzaC therapy was initiated in 9/2004. A hematologic improvement (HI) was achieved according to modified IWG criteria with marrow myeloblasts < 5%, RBC transfusion independence and a persistence of mild splenomegaly. A stable HI was maintained for 4 years on monthly AzaC therapy until relapse. At progression (10/2008), marrow myeloblasts increased to 11%, RBC transfusion requirement resumed, platelets fell to 20 × 109/L, the spleen enlarged and a new cytogenetically abnormal clone emerged, 46,XY,del(20)(q11q13),i(21)(q10),der(21)t(1;21)(q12;p11) resulting in trisomy for 1q. Administration of ON 01910.Na 1050 mg/m2/d x 72 hours CI was initiated 3 months after the last dose of AzaC. After 2 cycles of treatment evaluation demonstrated a reduction in: marrow myeloblast to 1%, spleen size by 50% and the MDS clone by 74% by standard cytogenetic analysis. Following the 4th and 7th cycles of treatment with ON 01910.Na, marrow myeloblasts ranged from 1 to 3%, palpable splenomegaly resolved, RBC transfusions decreased by 33% and platelets rose to 80 × 109/L. Although the conventional karyotype normalized after the 4th cycle, all 3 abnormalites were detected by FISH in 30% of cells following the 4th and 7th cycles suggesting a loss of proliferative capacity of the MDS clone. Patient 004 presented with high risk MDS, 19% marrow blasts, pancytopenia and a complex karyotype. AzaC treatment was initiated in 9/2002 leading to a stable HI, with marrow myeloblasts < 5%, RBC transfusion independence, and persistence of the MDS clone with no change in the complex karyotype, including deletion of the MLL locus. The HI was maintained for 6.5 years with monthly AzaC therapy until progression. At relapse marrow myeloblasts increased to 19%, and the complex karyotype persisted unchanged from initial diagnosis. ON 01910.Na treatment started (3/2009) and evaluations at the 2nd, 4th and 7th cycles revealed: a) reduction in % marrow myeloblasts to 12%, 8% and 7% respectively; b) persistence of abnormal karyotype in over 90% of metaphase and interphase cells, irrespective of the decreased % blasts; c) increased platelets from 20 to 40 × 109/L. Patient 002 and 004 remain on treatment with ON 01910.Na at 8+ and 5+ months, respectively. ConclusionsPreliminary data suggest that ON 01910.Na has an anti-proliferative modulatory effect on the MDS clone in pts progressing after AzaC therapy and should be explored in a broader MDS population. DisclosuresSilverman:Onconova Therapeutics: Research Funding. Off Label Use: ON01910Na is investigational and not FDA approved. Wilhelm:Onconova: Employment. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Holland:Onconova Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

WP1066 Disrupts Janus Kinase-2 and Induces Caspase-Dependent Apoptosis in Acute Myelogenous Leukemia Cells

Several cytokines and growth factors that stimulate the proliferation of acute myelogenous leukemia (AML) cells transduce their signals by activating the transcription factor Janus-activated kinase 2 (JAK2). Accordingly, the inhibition of JAK2 or of its downstream signaling pathways suppresses the proliferation of AML cells. Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G(0)-G(1) phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation.

Kinetin Riboside Is a Novel Targeted Inhibitor of CCND1 and CCND2 Transactivation with Substantial Preclinical Anti-Myeloma Activity.

Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood ; 106:296). To identify novel pharmaceutical inhibitors of cyclin D2 (CCND2) transactivation we therefore screened the Lopac, Prestwick and Spectrum libraries of drugs and natural compounds (n>4000) using NIH 3T3 cells stably expressing the CCND2 promoter driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Kinetin riboside, a nucleoside analogue and plant cytokinin hormone, as a novel inhibitor of CCND1+CCND2 transactivation. By immunoblotting, Kinetin riboside induced rapid suppression of cyclin D1 and D2 proteins (<6 hours) in H929, JJN3, Kms11 and U266 human myeloma cell lines (HMCL) that all over-express either cyclin D1 or D2 due to a range of transforming events relevant to myeloma (including deregulation of FGFR3, MMSET or c-Maf oncogenes, or translocation of CCND1 to the IgH locus). Similar results were obtained in primary CD138+ purified myeloma cells from 5/6 patients. To verify that cyclin D1 and D2 suppression induced by Kinetin riboside is a direct effect and does not occur secondary to cellular arrest in S-phase (when cyclin D protein levels decline) we examined the effects of Kinetin riboside on the cell cycle profile of HMCL. Within 20 hours Kinetin riboside caused the proportion of cells entering S-phase to fall by 50–70% in all HMCL tested, consistent with primary cyclin D suppression and secondary G0/G1 arrest. By MTT assay, Kinetin riboside is cytotoxic to HMCL with an IC50 of <1.7 mg/L (5uM) in 8/12 lines and <5mg/L (15uM) in 11/12 HMCL. By comparison, toxicity studies in Balb/c mice confirm that Kinetin riboside is tolerated in vivo at a dose of 80–100mg/kg i.p. or 25mg/kg i.v. Importantly, Kinetin riboside shows potent synergy with dexamethasone in HMCL that are poorly responsive to one or other agent and Kinetin riboside activity persists during co-culture with myeloma growth factors IL-6, IGF-1 and Baff. Moreover, when tested against unsorted patient bone marrow samples, Kinetin riboside preferentially killed CD138+ myeloma cells at up to 5–8 fold greater rate than normal marrow progenitors. Kinetin riboside induced myeloma cell death is mediated by apoptosis and is associated with caspase 9 cleavage and annexin V binding. Mechanistically, we show that kinetin riboside blocks CCND2 promoter transactivation induced by cAMP or by regulatory phosphoproteins (activated by Forskolin or the PP2A inhibitor, Cantharadin, respectively) and also blocks cis-activation of CCND1 induced by translocation to the IgH enhancer and trans-activation of CCND2 induced by cMaf or FGFR3 over-expression, indicating that kinetin riboside acts at a distal level to block transactivation of CCND1+CCND2 induced by multiple factors. Gene expression profiling reveals that KinR causes rapid induction of the transcriptional repressor, cAMP Response Element Modifier (CREM), which has been reported to bind the cyclin D2 promoter to regulate cyclin D2 expression (Muniz, 2006, Biol Reprod.), providing a putative mechanism for targeted suppression of CCND1+CCND2. Together these studies demonstrate a novel targeted mechanism and substantial pre-clinical activity for kinetin riboside and provide a rationale for clinical evaluation of this drug to improve the outcome of multiple myeloma.

Stromal Contact Inhibits Effects of Phorbol Esters but Does Not Inhibit Staurosporine Induced Megakaryocytic Differentiation in K562 Cells.

Development of human megakaryocytes is modulated at least in part by direct contact with stromal cells, however, the mechanisms that control this process have not been fully elucidated. Stromal contact is not needed for lineage commitment and direct contact of human progenitor cells with marrow stroma negatively influences megakaryocytic development. The K562 human erythroleukemia cell line has been extensively used as a model to study molecular aspects of megakaryocytic differentiation. Treatment of K562 with either phorbol-12 myristate-13 acetate (PMA), a PKC activator, or staurosporine (STSP) a PKC inhibitor, induces K562 cells to undergo differentiation to a megakaryocytic phenotype. Treated cells undergo growth arrest, changes in cellular morphology as well as increased expression of megakaryocytic/platelet-specific antigens, such as CD61. We have demonstrated that PMA-induced differentiation of K562 cells is inhibited by co-culture on the bone marrow stromal cell line, OP9. These cells remained small, continued to proliferate, and had less than 10% CD61 surface expression, similar to untreated K562. In contrast, K562 cells co-cultured on OP9 cells and treated with STSP retained the ability to differentiate. They were significantly larger than unstimulated K562 cells, exhibited megakaryocytic morphology, and had increased CD61 surface expression from 3.5±2.4% at baseline to 81±2.4%. These data suggest that while STSP and PMA recapitulate certain aspects of megakaryocytic differentiation, divergent signaling pathways may regulate the effects of stromal inhibition. Since stromal inhibition of differentiation plays an important role in the maintenance of hematopoietic progenitors in normal marrow, we investigated the molecular mechanisms involved in STSP induced differentiation. PMA induced megakaryocytic differentiation is dependent on sustained activation of ERK/MAPK, and stromal inhibition is associated with a loss of sustained Rap1 activation. The mechanism of action for STSP induced megakaryocytic differentiation has not been studied. Therefore we sought to investigate the ERK/MAPK signaling pathway during STSP induced differentiation. Using western blot analysis we found the small G protein c-RAF was phosphorylated at Ser338 when K562 cells were treated with either PMA or STSP, suggesting both agents activate c-RAF. In turn both led to a dramatic increase in ERK and p90RSK phosphorylation. We next examined the ERK signaling cascade during stromal inhibition. K562 cells co-cultured on OP9 cells and treated with PMA showed only a slight increase in c-RAF phosphorylation and no significant ERK or p90RSK phosphorylation, consistent with previous stromal inhibition data. However, STSP treatment of K562 co-cultured cells led to the robust phosphorylation of c-RAF and ERK. P90RSK was not significantly phosphorylated. Taken together these data suggest STSP treatment leads to activation of the ERK/MAPK pathway and that this activation may be Rap1 independent. Understanding the mechanisms involved in stromal inhibition and the divergent signaling pathways involved in megakaryocytic differentiation may bring to light novel functions of marrow stroma during normal and leukemic cell expansion.

Malignant progenitors from patients with CD87+ acute myelogenous leukemia are sensitive to a diphtheria toxin-urokinase fusion protein.

In previous studies, we demonstrated that the diphtheria toxin-urokinase fusion protein DTAT was selectively toxic to acute myeloid leukemia (AML) cell lines overexpressing the CD87 urokinase receptor. In the present study, we analyzed the sensitivity of patient leukemic progenitors to DTAT and correlated the sensitivity with CD87 expression. We isolated leukemic blasts by density gradient centrifugation and performed immunophenotyping by flow cytometry and blast sensitivity measurements by inhibition of cell proliferation and colony formation in semisolid media. We found CD87 overexpression in 18 (25%) of 71 patient leukemic blast samples, including 18 (28%) of 64 myeloid malignancies and 0 (0%) of 7 lymphoid malignancies. DTAT was toxic to patient leukemic blasts by both proliferation inhibition (IC50 <or=1 nM DTAT in 18/69 evaluable samples) and colony formation inhibition (>85% inhibition by 10 nM DTAT in 11/41 evaluable samples). Only AML and chronic myeloid leukemia (CML) blast crisis blasts (18/61 [30%]) were sensitive to DTAT by the proliferation inhibition assay. Lymphoid leukemia and chronic phase CML/chronic myelomonocytic leukemia (CMML) progenitors were insensitive to DTAT by the proliferation inhibition assay (n = 7 and n = 3, respectively). Similarly, normal marrow progenitors were insensitive to DTAT by both proliferation inhibition (n = 2) and colony inhibition (n = 5) assays. The DTAT toxicity measured by both proliferation inhibition assay and colony inhibition assay correlated with CD87 density (p < 0.0001 and p = 0.001, respectively). DTAT toxicity results were similar for leukemic blasts measured by either of the two assays (p = 0.0002). This study provides the first evidence that a urokinase receptor targeted diphtheria fusion protein is toxic to patient AML blasts. The work also suggests that blast proliferation assays yield similar responses to leukemia colony-forming cell colony assays.

The chemokine CCL21 protects normal marrow progenitors from Ara-C cytotoxicity.

Chemokines are a family of small proteins that regulate leukocyte infiltration into inflamed tissue and play key roles in the pathogenesis of many diseases. Some chemokines can also reversibly inhibit the proliferation of hematopoietic progenitors. We have previously found that the chemokine CCL21 (Exodus-2/SLC/6Ckine/TCA4) is a potent inhibitor of the proliferation of normal hematopoietic progenitors. In this study we sought to determine whether this inhibition of proliferation could be therapeutically exploited by protecting normal marrow progenitors from the cytotoxicity of the S phase-active chemotherapeutic agent Ara-C. Untreated and CCL21-pretreated mice were given doses of Ara-C that are toxic to marrow myeloid progenitors. The recovery of these myeloid progenitors was analyzed by colony formation assays. It was found that pretreatment with small doses of CCL21 prevented the death of normal murine marrow progenitors from the toxic effects of Ara-C. The chemokine CCL21 may be able to prevent Ara-C myelosuppression during acute leukemia induction chemotherapy, and thereby decrease morbidity and mortality of such therapy, and shorten hospital stays.

Ex vivo pharmacological purging of leukapheresis collections with nitrogen mustard: Amifostine pretreatment improves both early and late peripheral blood progenitor cell recovery

Ex vivo pharmacological purging of bone marrow has been used to eliminate clonogenic tumor cells contaminating the autograft and potentially responsible of relapse. A considerable improvement of pharmacological purging would be achieved only if normal marrow progenitor cells could be selectively protected by the cytotoxicity of these agents. Amifostine (WR-2721; Ethyol), a phosphorylated aminothiol compound, has been shown to have this property both in vivo and in vitro. We describe here, an experimental model for ex vivo purging of peripheral blood progenitor cell (PBPC) collections based on the combination of 3 mg/ml of amifostine and the alkylating agent nitrogen mustard. Amifostine pretreatment resulted in a statistically significant protection of normal late and early progenitor cells. Under the same experimental conditions, we observed a 4–6 log reduction of contaminating leukemic cells (i.e., K-562 and CEM) and in contrast to the protection of normal peripheral blood progenitor cells, preincubation of contaminating K-562 or CEM with amifostine did not significantly alter the LD95 nitrogen mustard concentration. Moreover, when we tested fresh human leukemia progenitor cells, amifostine pretreatment sensitized the leukemic cells to the cytotoxic effects of NM.

A role for paclitaxel in the combination chemotherapy of acute myeloblastic leukaemia: preclinical cell culture studies.

Paclitaxel dose responses in culture have been investigated alone and in association with cytosine arabinoside (ARA-C) and all-trans retinoic acid (ATRA), with the objective of identifying a role for paclitaxel in the treatment of acute myeloblastic leukaemia (AML). Initial studies were done to determine if paclitaxel dose responses of AML blast cell precursors were altered by regulatory compounds known to modify the dose responses of ARA-C. In contrast to ARA-C, paclitaxel dose responses were independent of cell culture method, the growth factors G-CSF and GM-CSF, and the ligands all-trans retinoic acid (ATRA) and hydrocortisone. Most blast cell populations were sensitive to paclitaxel; compared with normal marrow progenitors the dose responses were markedly heterogenous with some more, and others less, sensitive. Remission marrow progenitor paclitaxel responses resembled those of AML blasts in heterogeneity. The cell culture model tested the effect of pacliataxel and ATRA on the ARA-C dose responses of OCI/ AML-5; paclitaxel exposure was either before or after ARA-C to test for an effect of schedule; ATRA was added to the MEC cultures after paclitaxel and ARA-C. Repeat experiments were done to test three dose levels each of paclitaxel and ATRA. When paclitaxel was given after ARA-C, synergism was found for all but one of the dose combinations tested; only three examples of synergy were seen when paclitaxel preceded ARA-C. The studies justify trials combining ARA-C, paclitaxel and ATRA using a schedule suggested by the cell culture findings.

The development and magnitude of thermotolerance during chronic hyperthermia in murine bone marrow granulocyte-macrophage progenitors: I.

Murine bone marrow granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during exposure to temperatures < 42.5 degrees C. Bone marrow from the tibia and femora was heated to 40-42 degrees C (i.e. chronic hyperthermia), and challenged immediately with 15 min at 44 degrees C at regular intervals during treatment (step-up heating). CFU-GM were heated and cultured in McCoy's 5A medium + 15% FBS (fetal bovine serum) and lung-conditioned medium (source of colony stimulating factor) in semisolid agar. The kinetics of thermotolerance development and decay, and the magnitude of the thermotolerance during chronic heating with temperatures of 40-41.5 degrees C were similar. Survival increased rapidly to a maxima by approximately 120 min of hyperthermia (temperatures of 40-41.5 degrees C) and thereafter decreased with a slope similar to the controls. Normalization for cell killing by chronic hyperthermia that occurred during "step-up' heating permitted analysis of thermotolerance in the surviving cells. The surviving fraction after 15 min at 44 degrees C, during incubation at 40, 41 and 41.5 degrees C increased from 0.13 to maxima of 0.56 +/- 0.04, 0.71 +/- 0.03 and 0.82 +/- 0.03 respectively, by 150 min and did not decrease for up to 480 min during chronic hyperthermia. The surviving fraction after 15 min at 44 degrees C during incubation at 42 degrees C increased more slowly than during incubations at 40-41.5 degrees C. The survival of thermotolerant cells after exposure to 15 min at 44 degrees C during 42 degrees C chronic hyperthermia was maximal at 0.87 +/- 0.08 by 120 min and then decreased after approximately 150 min of exposure to 42 degrees C. The thermotolerance ratios (TTR's) were 4.0, 5.4, 6.7 and 6.9 for temperatures of 40, 41, 41.5 and 42 degrees C respectively. The results suggest that chronic hyperthermia temperatures (i.e. 40-42 degrees C) induce rapid thermotolerance development in CFU-GM during the thermal exposure and protect this normal marrow progenitor during whole body hyperthermia or ex vivo purging of leukaemic cells.

Effect of Interleukin-1β Converting Enzyme Inhibitor on Acute Myelogenous Leukemia Progenitor Proliferation

AbstractInterleukin-1β (IL-1β) converting enzyme (ICE) is a cysteine protease that specifically cleaves precursor IL-1β to its biologically active form. Recent studies have also implicated ICE in the induction of apoptosis in vertebrate cells. Because IL-1 plays a major role in acute myelogenous leukemia (AML) blast proliferation, we sought to investigate the effect of ICE inhibition on AML progenitors. To do this, we used boc-aspartyl (benzyl) chloromethylketone (BACMK) an inhibitor designed to penetrate cells and bind covalently to the active site of ICE. Our preliminary experiments showed that incubation of activated peripheral blood cells with 2.5 μmol/L of BAMCK downregulated production of mature IL-1β but had no effect on tumor necrosis factor-α. To test the effects of the inhibitor on AML cells, we first used the OCI/AML3 cell line. We found that these cells produce IL-1β and bind the biotinylated cytokine and that IL-1 inhibitors, such as IL-1 neutralizing antibodies, IL-1 receptor antagonist, and soluble IL-1 receptors, specifically inhibit OCI/AML3 proliferation, indicating that IL-1β is an autocrine growth factor for OCI/AML3 cells. The ICE inhibitor suppressed OCI/AML3 growth in a dose-dependent manner (at 0.4 to 4 μmol/L) and downregulated mature IL-1β production, as assessed by Western immunoblotting. Similar results were obtained with marrow aspirates from 16 AML patients. The ICE inhibitor suppressed proliferation of AML precursors (by up to 78%; mean, 44%) in a dose-dependent fashion at concentrations ranging from 0.4 to 5 μmol/L but not proliferation of normal marrow progenitors; the suppressive effect was reversed by IL-1β. Furthermore, incubation of AML cells with 4 μmol/L BAMCK downregulated the production of mature IL-1β, suggesting that the growth-inhibitory effect is mediated through suppression of the biologically active cytokine. Our data indicate that inhibition of ICE suppresses AML blast proliferation and suggest that ICE inhibitors may have a role in future therapies for AML.

1118 Amifostine improves the antileukemic therapeutic index of mafosfamide: Implications for bone marrow purging

To test the potential use of amifostine (Ami) to protect normal bone marrow (N1 BM) progenitor cells during ex vivo purging of leukemia, the dose response of mafosfamide (Mfs) ± Ami on N1 BM progenitor cells was determined and the LD95 was calculated. Ami pretreatment resulted in a statistically significant protection of CFU-GM and erythroid blast-forming units (BFU-E) from Mfs toxicity. The mean ± SEM μg/ml LD95 concentrations were: CFU-GM, Mfs alone 54 ± 13 vs Ami-Mfs 66 ± 14, p. 005, BFU-E, Mfs alone 48 ± 14, vs Ami-Mfs 61 ± 11, p .005. In contrast, Ami pretreatment sensitized CFU-L to Mfs toxicity: LD95 for Mfs alone 33 ± 4 vs Ami-Mfs 27 ± 3, p .003. At the LD95 fur CFU-GM and BFU-E, Mfs alone resulted in a ≈4 log cell-kill for L-CFU. Ami protection of N1 BM resulting in a higher Mfs LD95 concentration, coupled with the Ami-sensitization of the leukemia, provided an estimated additional 6+ log leukemia cell-kill. This enhanced selective leukemia cell-kill from Mfs during ex vivo purging using the LD95 concentration of Mfs for normal marrow progenitors offers the potential for lowering the incidence of leukemic relapse while preserving a greater number of normal progenitor cells for engraftment following autologous transplantation.