Inhibition of Acute Leukemia By a Small Molecule KYA1797K Which Destabilize RAS and β-Catenin

Introduction: Signaling pathways in acute leukemia are aberrantly activated to cause leukemogenesis and relapse after treatment. Like in many other malignancies, upregulation of WNT/beta-catenin pathway and hyperactive RAS is known to be associated with treatment resistance in leukemia. However, there are little studies about RAS singaling pathyway and leukemia, and it is a field of study that needs to be revealed. KYA1797K is a recently developed small molecule, binds directly to RGS domain of axin and enhances the beta-catenin destruction complex which activates GSK3beta and results in degradation of beta-catenin and RAS. In the current study, we tried to find the role of RAS inhibition by KYA1797K in leukemic cell lines and in patient's BM samples. Moreover, other small molecule PCK412 (Midosaturin) was also used for comparison. Materials & Methods: Leukemic cells (MOLT-4, THP-1, MOLT-4, Jurka, KG-1, MV4-11, RS4-11) were cultured in RPMI1640 media under various concentration (0.1-10µM for KYA1797K, 0.5-500nM for PKC412) for 48h and with Erlotinib (1µM) for comparison. Cell proliferation assay on each leukemic cell was done and immunoblotting for β-catenin, GSK3β, Pan-RAS, N-RAS was checked. Downstream targets of Wnt pathway (c-Myc, CD44, LEF1, Met, TCF1/TCF7) were studied by immunoblotting. MOLT-4 was stimulated with Wnt3a (200ng/mL, 4h) and changes in Wnt pathway were observed. Bone marrow samples of AML and ALL patients were evaluated for β-catenin and RAS. KYA1797K (Nat Chem Biol 2016, 12:593) was kindly provided by Prof. Kang-Yell Choi. Results: Suppression of leukemic cells by KYA1797K was evident starting from the concentration of 5 microM. (fig.1) Beta-catenin was down regulated in all cell lines by KYA1797K. Pan-RAS decreased in MOLT-4 and THP-1. All the downstream targets evaluated were down regulated by KYA1797K in MOLT-4 culture, and was evident at the concentration of 5microM. (fig.2) Stimulation of Wnt pathway by Wnt3a was inhibited by KYA1797K. (fig.3) Bone marrow samples from 7 ALL patients showed various status of β-catenin and RAS expression. Two high-risk patients showed suppression of β-cateinin and N-RAS by KYA1797K. (fig.4) In MV4-11 (FLT3 mutant) and RS4-11 (FLT3 wild type), IC50 for PKC412 was higher in RS4-11 compared to MV4-11 while KYA1797K showed same IC50 in both cell lines. β-catenin and RAS downregulation was observed by KYA1797K. Effects of KYA1797K analyzed by qRT-PCR and immunoblot for FLT3, N-RAS, MEK, ERK, ETS2 showed that KYA1797K downregulates FLT3 in MV4-11 even though FLT3 is not the main target of action. It was less effective on RS4-11. (fig.5) In bone marrow samples of ALL patient with FLT3 mutation, KYA1797K 1µM showed effect in reducing leukemia cells. (fig.6) Conclusion: This preclinical study suggests that KYA1797K may be an option for patients with acute leukemia. KYA1797K effectively destabilized β-catenin and RAS in acute leukemia even under Wnt pathway activation. FLT3, N-RAS, MEK, ERK, ETS2 were down regulated by KYA1797K, hence KYA1797K has a potential application for acute leukemia with FLT3 mutation. Extended studies including further in vivo study are needed to build up a strategy in small molecule therapy to target RAS in acute leukemia. Although with limitation, we suggest RAS inhibitor as a potential drug for leukemia. Disclosures No relevant conflicts of interest to declare.

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Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽

10.1182/blood.v65.1.100.100 ◽

1985 ◽

Vol 65 (1) ◽

pp. 100-106 ◽

Cited By ~ 9

Author(s):

HN Steinberg ◽

AS Tsiftsoglou ◽

SH Robinson

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Colony Formation ◽

Leukemic Cell ◽

K562 Cells ◽

Leukemic Cells ◽

Cell Phenotype ◽

Normal Bone Marrow ◽

Normal Bone ◽

Leukemic Cell Lines

Abstract The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

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Modification of Gene Expression in Mesenchymal Stromal Cells of the Leukemia Patients during Chemotherapy

Blood ◽

10.1182/blood.v128.22.5065.5065 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5065-5065

Author(s):

Tamara Sorokina ◽

Irina Shipounova ◽

Alexey Bigildeev ◽

Nina I. Drize ◽

Larisa A. Kuzmina ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Leukemia ◽

Fetal Calf Serum ◽

Calf Serum ◽

Leukemic Cell ◽

Expression Level ◽

Leukemic Cells ◽

Disease Manifestation ◽

Relative Expression Level

Abstract Background In patients with acute leukemia the stromal microenvironment is deeply modified. Disturbances in signaling pathways, genetic abnormalities and functional changes in mesenchymal cells of these patients have been previously described. Chemotherapy also affect stromal progenitor cells. A damaged microenvironment might impair hematopoiesis in acute leukemia patients. Aims To investigate the relative expression level in MMSCs and CFU-Fs, derived from the bone marrow (BM) of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients before and over the course of chemotherapy. Methods 54 newly diagnosed cases (33 AML, 21 ALL) were involved in the study after informed consent. BM was aspirated prior to any treatment (time-point 0) and at days 37, 100 and 180 since the beginning of treatment of acute leukemia. MMSCs were cultured in aMEM with 10% fetal calf serum, CFU-Fs, in aMEM with 20% fetal calf serum. The relative expression level (REL) of different genes was measured by TaqMan RQ-PCR. As a control MMSCs and CFU-Fs from 88 healthy donors were used. Results At the time of the disease manifestation the analysis of gene expression in MMSCs from acute leukemia patients revealed a significant increase in the REL of genes which regulate immune system responses and thereby can influence on the leukemic cell proliferation and migration (IL-6, IL-8, IL-1b and IL-1R1) (Pic.1). Also at the time of the diagnosis an increase in REL of genes, that are responsible for hematopoiesis regulation, was observed. For example, the REL of CSF1 that can influence on leukemic cells proliferation was increased at the disease manifestation and became normal during the treatment. The same dynamics was observed in the REL of JAG1 that has an antiapoptotic effect on leukemic cells. The REL of LIF had been also significantly increased at the disease manifestation, reflecting the efforts of MMSCs to inhibit leukemic proliferation. Chemotherapy affected REL of the studied genes differently. The treatment lead to the downregulation of IGF, TGFB1 and TGFB2 (Pic.2). As far asTGFB1 and 2 inhibit the differentiation of mesenchymal stem cells, and IGF is associated with myelodysplastic changes in elderly bone marrow, so their downregulation may refer to the effectiveness of therapy. The REL of genes regulating MMSC proliferation (PDGFRa and PDGFRb, FGF2, FGFR1 and 2) increased during chemotherapy. Exploring cell adhesion molecules, the decrease in the REL of their encoding genes was observed. As far as VCAM facilitate the leukemic cell extravasation and ICAM was shown to depress the Th17 cell differentiation, the down-regulation of their genes may reflect the microenvironment restoration. The influence of chemotherapy lead to decrease in REL of genes, associated with MMSCs differentiation (BGLAP and SOX9 (Pic.3)), reflecting the mechanism of the blocking of MMSCs migration and differentiation under the stress conditions. The alterations of bone marrow stroma were more pronounced in patients who didn't achieve remission. The REL of 9 genes was studied in CFU-F colonies. There were no differences in gene expression in CFU-Fs before the treatment, except for an increase in the REL of PPARg in acute leukemia CFU-Fs. During the treatment, a decrease in the REL of SPP1 and an increase in the REL of FGFR1 and 2 were observed. Conclusion Therefore, chemotherapy used does not impair the functional ability of MMSCs and CFU-Fs, but influence on their gene expression profile. The two types of precursors are affected differently, indicating their different differentiation level and functions. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

Blood ◽

10.1182/blood.v67.3.802.802 ◽

1986 ◽

Vol 67 (3) ◽

pp. 802-804 ◽

Cited By ~ 39

Author(s):

Y Moriyama ◽

M Narita ◽

K Sato ◽

M Urushiyama ◽

S Koyama ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Progenitor Cells ◽

Autologous Bone Marrow Transplantation ◽

Leukemic Cell ◽

Autologous Bone Marrow ◽

Leukemic Cells ◽

Neoplastic Disease ◽

Myeloid Progenitor Cells

Abstract The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.

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Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽

10.1182/blood.v65.1.100.bloodjournal651100 ◽

1985 ◽

Vol 65 (1) ◽

pp. 100-106

Author(s):

HN Steinberg ◽

AS Tsiftsoglou ◽

SH Robinson

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Colony Formation ◽

Leukemic Cell ◽

K562 Cells ◽

Leukemic Cells ◽

Cell Phenotype ◽

Normal Bone Marrow ◽

Normal Bone ◽

Leukemic Cell Lines

The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

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Utilization of a colony assay to assess the variables influencing elimination of leukemic cells from human bone marrow with monoclonal antibodies and complement

Blood ◽

10.1182/blood.v65.4.945.bloodjournal654945 ◽

1985 ◽

Vol 65 (4) ◽

pp. 945-950

Author(s):

TW LeBien ◽

DE Stepan ◽

RM Bartholomew ◽

RC Stong ◽

JM Anderson

Keyword(s):

Bone Marrow ◽

Monoclonal Antibodies ◽

Cell Lines ◽

Bone Marrow Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Colony Assay ◽

Leukemic Cell Lines ◽

Human Leukemic Cells ◽

Cell Colony

We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB- NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.

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ASPP2 Expression In Acute Leukemia Modulates Response to Therapy

Blood ◽

10.1182/blood.v116.21.3143.3143 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3143-3143

Author(s):

Kerstin M Kampa-Schittenhelm ◽

Charles D Lopez ◽

Marcus Schittenhelm

Keyword(s):

Tumor Suppressor ◽

Acute Leukemia ◽

Cell Lines ◽

Induction Chemotherapy ◽

Small Molecule ◽

Response To Therapy ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Leukemia Cells ◽

P53 Pathway

Abstract Abstract 3143 Acute myeloid leukemias (AML) remain difficult to treat and therapy outcome is far from satisfactory for most disease subgroups. Inactivation of the p53 tumor suppressor pathway by mutation is a frequent event in many cancers that promotes tumorigenesis and resistance to chemotherapy. Although p53 mutations are rare in AML it is not well studied whether the p53 pathway is influenced by other mechanisms instead. ASPP2 (Apoptosis Stimulating Protein of p53 2) is a highly regulated member of a family of p53-binding proteins that enhance apoptosis at least in part through stimulation of p53-transactivation of selected pro-apoptotic target genes. We previously demonstrated in a mouse model that ASPP2 is a haploinsufficient tumor suppressor (Kampa et al., PNAS 2009), and low ASPP2 expression levels have been associated with aggressive courses of different tumors such as breast cancer and lymphoma. We have now studied how ASPP2 expression correlates with response to therapy using in vitro models as well as patient-derived acute leukemia cells collected pre- and post- chemotherapy. We first analyzed changes in ASPP2 protein expression after treatment with chemotherapy or small molecule tyrosine-kinase inhbitors. We found that ASPP2-induction was cell-type specific in various established leukemia lines including Jurkat and HL60 (ASPP2 levels induced) and MOLM14 cells (ASPP2 levels unchanged). To test if ASPP2 levels modulated growth or response to therapy of leukemia cells, we generated three different ASPP2-siRNA constructs and transiently introduced them by lipofection into various lymphoid and myelogenous leukemia cell lines, including K562, Kasumi1, HL60, MOLM14 and Jurkat. After ASPP2 silencing, we observed, with the exception of MOLM14 cells, an up to 3-fold increase in cell proliferation measured by an XTT-assay compared to an empty vector control. We also treated siRNA-silenced K562, Kasumi1, HL60 and Jurkat cell lines with daunorubicin or small molecules targeting cell line-specific mutations in FLT3, KIT or ABL. Cell lines with attenuated ASPP2 expression displayed a significantly (∼50%) lower rate of apoptosis-induction in AnnexinV-assays after chemotherapy as well as small molecule inhibitor treatment as compared to a negative control. Interestingly, treated and siRNA-silenced leukemic cells frequently demonstrated enlarged morphology consistent with mitotic catastrophe. To study ASPP2 expression in humans, we quantified ASPP2 levels by qRT-PCR and intracellular immunophenotyping in circulating leukemic cells derived from patients at several timepoints before and during induction-chemotherapy (n=63). We found that pre-treatment ASPP2 basal levels were variable in acute leukemias. Additionally, we found that induction-chemotherapy increased ASPP2 expression in leukemic cells in a subgroup of patients. Univariate and multivariate analysis of the correlation of available clinical data and patient outcomes with ASPP2 expression in these patient datasets is ongoing. Taken together, our results demonstrate that dysfunctional regulation of ASPP2 expression may contribute to the biology of leukemogenesis and to primary therapy resistance in a subgroup of patients with acute leukemia. This data provides important and clinically relevant insight into how the p53 pathway can be inactivated in acute leukemia and opens new avenues for investigation. Prospective clinical studies are warranted in order to further define the role of the ASPP2 pathway as a therapeutic target and as biomarker for response to therapy. Disclosures: No relevant conflicts of interest to declare.

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Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines

Blood ◽

10.1182/blood.v70.1.192.bloodjournal701192 ◽

1987 ◽

Vol 70 (1) ◽

pp. 192-199 ◽

Cited By ~ 1

Author(s):

B Lange ◽

M Valtieri ◽

D Santoli ◽

D Caracciolo ◽

F Mavilio ◽

...

Keyword(s):

Growth Factor ◽

Acute Leukemia ◽

Cell Lines ◽

Leukemic Cell ◽

Media Analysis ◽

Leukemic Cells ◽

Gm Csf ◽

Leukemic Cell Lines ◽

Dependent Cell ◽

Childhood Acute Leukemia

Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony- stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM- CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.

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Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

Blood ◽

10.1182/blood.v67.3.802.bloodjournal673802 ◽

1986 ◽

Vol 67 (3) ◽

pp. 802-804

Author(s):

Y Moriyama ◽

M Narita ◽

K Sato ◽

M Urushiyama ◽

S Koyama ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Progenitor Cells ◽

Autologous Bone Marrow Transplantation ◽

Leukemic Cell ◽

Autologous Bone Marrow ◽

Leukemic Cells ◽

Neoplastic Disease ◽

Myeloid Progenitor Cells

The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.

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The Persistence of Extramedullary Leukemic Infiltrates during Bone Marrow Remission of Acute Leukemia

Blood ◽

10.1182/blood.v26.2.133.133 ◽

1965 ◽

Vol 26 (2) ◽

pp. 133-141 ◽

Cited By ~ 101

Author(s):

BOYD A. NIES ◽

GERALD P. BODEY ◽

LOUIS B. THOMAS ◽

GEORGE BRECHER ◽

EMIL J. FREIREICH

Keyword(s):

Central Nervous System ◽

Bone Marrow ◽

Nervous System ◽

Acute Leukemia ◽

Lymph Nodes ◽

Leukemic Cell ◽

Leukemic Cells ◽

Common Site ◽

The Central Nervous System

Abstract Leukemic cell infiltrates were found at autopsy in the tissues of 10 of 15 patients with acute leukemia dying during "complete bone marrow remission." The kidney was the most common site of leukemic cell infiltrates followed by the liver, testes, bowel, lung, central nervous system, and lymph nodes. These findings indicate that leukemic cells are not completely eradicated by current chemotherapy even in patients in whom no leukemic cells can be identified in the bone marrow. The distribution of residual leukemic cells demonstrates that the central nervous system is not the only reservoir of leukemic cells in patients during bone marrow remission.

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Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia

Blood ◽

10.1182/blood.v71.1.166.bloodjournal711166 ◽

1988 ◽

Vol 71 (1) ◽

pp. 166-172

Author(s):

ML Auber ◽

LJ Horwitz ◽

A Blaauw ◽

S Khorana ◽

S Tucker ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Leukemic Cell ◽

Leukemic Cells ◽

Lymphoid Leukemia ◽

Colony Forming Units ◽

Cell Kill ◽

Macrophage Colony ◽

Vitro Effect

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4- hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.

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