SHIP1/SH2 domain-containing inositol-5′-phosphatase 1 (IC50 = 2.5 μM)

3α-Aminocholestane (3AC) therapy substantially reduces OPM2 cell viability. When compared to OPM2 cells, RPMI8226 and U266 cells exhibit much lower sensitivity to 3α-Aminocholestane treatment; yet, viability is significantly reduced at doses of ≥12.5 μM. After being treated for 36 hours with 3α-Aminocholestane, the proportion of cells in the S phase is significantly decreased, and the number of cells in the G2/M phase increases. On the other hand, in the less proliferative RPMI8226 and U266 cells, treatment with 3α-Aminocholestane blocks cell cycle progression in the G0 /G1 phase and results in a lower percentage of cells progressing through the S phase[2].

Upon OPM2 challenge, it is discovered that 3α-Aminocholestane (3AC) leads to decreased multiple myeloma (MM) growth in vivo, as measured by the amount of free human Igλ light chain in the plasma. Furthermore, peripheral blood from mice treated with 3-aminocholestane shows less circulating OPM2 cells, as detected by human HLA-ABC labeling, as compared to vehicle controls. Most notably, mice treated with 3α-Aminocholestane had much improved survival rates following tumor challenge. When mice treated with 3α-Aminocholestane fail to respond to treatment, it is discovered that MM tumors have an overexpression of SHIP2, which is similar to when OPM2 cells are treated in vitro and implies that tumor cells with higher SHIP2 expression may be chosen by SHIP1 inhibition[2].

Detection of Phosphatase Enzymatic Activity [2]
Fluorescent polarization assay was used as described previously. In short, recombinant SHIP1 or SHIP2 is mixed with its substrate PtdIns(3,4,5)P3 in the presence of potential chemical inhibitors. The reaction product is mixed withPtdIns(3,4)P2 detector protein and a fluorescent PI(3,4)P2 probe. Newly synthesizedPtdIns(3,4)P2 displaces the detector protein, thereby enhancing an unbound fluorescent probe in the mixture and decreasing mean polarization units. Thus, identified SHIP inhibitors, (2-phenyl-benzo[h]quinolin-4-yl)-[2]piperidyl-methanol hydrochloride (1PIE), 1-[(chlorophenyl)methyl]-2-methyl-5-(methylthio)-1H-indole-3-ethanamine hydrochloride (2PIQ) and (2-adamantan-1-yl-6,8-dichloro-quinolin-4-yl)-pyridin-2-yl-methanol hydrochloride (6PTQ) were subsequently tested for inhibition of free phosphate production by recombinant SHIP1 or SHIP2 by Malachite Green assay as described before or by fluorescent polarization assay. To demonstrate selectivity of the compounds for SHIP1 and SHIP2 over other phosphatases, SHIP1 and the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) were immunoprecipitated from OPM2 cells. For this purpose, OPM2 cells were lysed in IP-lysis buffer (20 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton × 100, 1 mmol/L phenylmethylsulfonyl fluoride and Halt protease inhibitor), and SHIP1 or OCRL were immunoprecipitated by using mouse IgG antibodies. Beads were washed four times with immunoprecipitation (IP) lysis buffer and once with Tris-buffered saline (TBS)/MgCl2 (10 mmol/L) and resuspended in TBS/MgCl2. SHIP inhibitors (200 μmol/L) were added to the beads for 5 min, after which immunoprecipitated SHIP1 was incubated in the presence of 100 μmol/L PtdIns(3,4,5)P3, whereas immunoprecipitated OCRL was incubated in the presence of 100 μmol/L PtdIns(4,5)P2 for 30 min. Malachite Green solution was added according to the manufacturer’s instructions, and the plate was read after 20 min. Identification of 3α-aminocholestane (3AC) was described previously

Cell Viability Assay [2]
Cells were treated in triplicate or more with increasing concentrations of compounds. Cell viability was determined with a Cell Counting Kit per the manufacturer’s instructions. The odds density (OD) of compound-treated cells was divided by the OD of their vehicle control, and the viability was expressed as a percentage of untreated cells. Results are expressed as mean ± standard error of the mean (SEM) of three individual experiments. For PIP add-back experiments, MCF-7 cells were treated for 2 h with 10 μmol/L SHIP inhibitors, after which cells were washed and fresh medium was added. Cells were subsequently cultured in the absence (0 μmol/L) or presence (10 or 20 μmol/L) of eitherPtdIns(3,4)P2-diC16 (P-3416) or PtdIns(3,5)P2-diC16 (P-3516) for 36 h, after which cell viability was determined by the Dojindo Cell Counting Kit.

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Cell viability assay [1]
100,000 human ALL cells were seeded in a volume of 50 μl medium in one well of a 96-well plate. Imatinib or any other inhibitor was diluted and incubated at the indicated concentration in a total volume of 100 μl medium. After 3 days, cell counting kit-8 was used to determine the number of viable cells. Fold changes were calculated using baseline values of vehicle treated cells as a reference (set to 100%).

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Flow cytometry [1]
Antibodies used in flow cytometry are mentioned in Supplementary Table 6. For cell-cycle analysis, the BrdU flow cytometry kit or Click-iT EdU Flow Cytometry Assay Kit was used according to the manufacturer’s instructions. For evaluation of intracellular ROS levels, ALL cells were incubated for 7 min with 1 μM 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) at 37°C for oxidation of the dye by ROS. After washing with PBS, the cells were incubated additional 15 min at 37°C in PBS to allow complete deacetylation of the oxidized form of CM-H2DCFDA by intracellular esterases. The levels of fluorescence were then directly analyzed by flow cytometry, gated on viable cells.

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Western blotting [1]
CelLytic buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail set II were used to lyse cells. 10 μg of protein lysates per sample were separated on mini precast gels and transferred on nitrocellulose membranes For the detection of proteins, primary antibodies, alkaline-phosphatase conjugated secondary antibodies, and chemiluminescent substrate were used. Details of primary antibodies were shown in Supplementary Table 7.

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Colony forming assay for mouse cells [1]
10,000 BCR-ABL1-transformed ALL cells or 100,000 CML-like cells were used for this assay. Cells were resuspended in murine MethoCult medium and plated on dishes (3 cm in diameter) with an extra dish of water to prevent evaporation. After 7 to 14 days, colonies were counted.

OPM2 Tumor Challenge Studies [2]
NOD/SCID/γcIL2R (NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were injected intraperitoneally with 1 × 107 OPM2 cells and 6 h later received an initial injection of 3α-aminocholestane (3AC)  or vehicle. 3α-aminocholestane (3AC)  was suspended in a 0.3% Klucel/H2O solution at 11.46 mmol/L and administered by intraperitoneal injection of 100-μL solution. Vehicle-treated mice received 100-μL injection of 0.3% Klucel/H2O solution. The final concentration of 3α-aminocholestane (3AC)  in the treated mice was 60 μmol/L. The mice were then treated with 3α-aminocholestane (3AC)  or vehicle daily for the next 6 d and then twice per week in the remaining 15 wks of the survival study. In some instances, tumors from the vehicle- or 3α-aminocholestane (3AC)  -treated hosts were excised and single-cell suspensions were made for Western blot analysis of SHIP2 expression after mice were deemed to be moribund and recommended for humane euthanasia by veterinary staff.

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Enzyme-Linked Immunosorbent Assay for Human Igλ Light Chain in Mouse Peripheral Blood [2]
Mice were bled into a serum collection tube 4 wks after the OPM2 challenge, and serum was obtained after pelleting of blood cells at 5,000g for 5 min. Human Igλ light chain amounts were determined using an Ig light chain detection kit from Biovendor per the manufacturer’s instructions.

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Detection of Circulating OPM2 Cells in Mouse Blood [2]
Mice were bled into a blood collection tube 4 wks after OPM2 challenge and red cells were lysed. White blood cells were incubated with anti-CD16/32 to block Fc receptor binding and then stained with antibodies against human HLA-ABC, clone W6/32. Samples were acquired on an LSRII cytometer (Becton Dickinson), and dead cells were excluded from the analysis after cytometer acquisition by exclusion of cells that stained positively for DAPI (di aminido phenyl indol).

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3α-Aminocholestane is suspended in a 0.3% Klucel/H2O solution at 11.46 mM and administered by intraperitoneal injection of 100 μL solution.

NOD/SCID/γcIL2R (NSG) mice

[1]. Signalling thresholds and negative B-cell selection in acute lymphoblastic leukaemia. Nature. 2015 May 21;521(7552):357-61.

[2]. Therapeutic Potential of SH2 Domain-Containing Inositol-5′-Phosphatase 1 (SHIP1) and SHIP2 Inhibition in Cancer. Mol Med. 2012 Feb 10;18:65-75.

B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
A small molecule inhibitor against INPP5D, 3-α-aminocholestane, 3AC (Extended Data Fig. 10f) selectively inhibited enzymatic activity of INPP5D (SHIP1; IC50 ~2.5 μmol/l) but not related phosphatases INPP5L1 (SHIP2) and PTEN (IC50 >20 μmol/l). Treatment of patient-derived Ph+ ALL cells with 3AC induced strong hyperactivation of SYK (Fig. 4a). In patient-derived myeloid CML samples, baseline levels of Syk activity were very low and not responsive to 3AC treatment (Extended Data Fig. 10g). Biochemical characterization of 3AC-mediated inhibition of INPP5D in patient-derived Ph+ ALL cells revealed potent and transient hyperactivation of proximal pre-BCR signaling molecules (Fig. 4a). Treatment of patient-derived TKI-resistant Ph+ ALL cells with 3AC induced cell death within four days. Importantly, pre-treatment of Ph+ ALL cells with the SYK-inhibitor (PRT06207) largely protected Ph+ ALL cells against 3AC-induced cell death (Fig. 4b), demonstrating that hyperactivation of Syk is required for induction of cell death. Dose-response analyses revealed that 3AC is selectively toxic for patient-derived Ph+ ALL cells (IC50=2.8 μmol/l; n=5) compared to mature B cell lymphoma (n=5; Extended Data Fig. 10h). We next studied drug-responses in a panel of six cases of Ph+ ALL from patients who relapsed under TKI-therapy, including three cases with global TKI-resistance owing to the BCR-ABL1T315I mutation. As expected, treatment with the TKI Imatinib had no effect in BCR-ABL1T315I cases (Extended Data Fig. 10i). In contrast, 3AC induced massive cell death (>95%) in all six cases of Ph+ ALL regardless of BCR-ABL1 mutation status (Extended Data Fig. 10i). Likewise, treatment of NOD/SCID transplant recipient mice carrying TKI-resistant patient-derived (BCR-ABL1T315I) Ph+ ALL cells with 3AC significantly prolonged overall survival (P=0.0002, log rank test; Fig. 4c) and reduced leukemia burden (Fig. 4d). While further studies are needed to optimize pharmacological targeting of this pathway, these experiments identify transient hyperactivation of SYK and engagement of negative B cell selection as a powerful new strategy to overcome drug-resistance in Ph+ ALL. [1]

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Many tumors present with increased activation of the phosphatidylinositol 3-kinase (PI3K)-PtdIns(3,4,5)P(3)-protein kinase B (PKB/Akt) signaling pathway. It has long been thought that the lipid phosphatases SH2 domain-containing inositol-5'-phosphatase 1 (SHIP1) and SHIP2 act as tumor suppressors by counteracting with the survival signal induced by this pathway through hydrolysis or PtdIns(3,4,5)P(3) to PtdIns(3,4)P(2). However, a growing body of evidence suggests that PtdInd(3,4)P(2) is capable of, and essential for, Akt activation, thus suggesting a potential role for SHIP1/2 enzymes as proto-oncogenes. We recently described a novel SHIP1-selective chemical inhibitor (3α-aminocholestane [3AC]) that is capable of killing malignant hematologic cells. In this study, we further investigate the biochemical consequences of 3AC treatment in multiple myeloma (MM) and demonstrate that SHIP1 inhibition arrests MM cell lines in either G0/G1 or G2/M stages of the cell cycle, leading to caspase activation and apoptosis. In addition, we show that in vivo growth of MM cells is blocked by treatment of mice with the SHIP1 inhibitor 3AC. Furthermore, we identify three novel pan-SHIP1/2 inhibitors that efficiently kill MM cells through G2/M arrest, caspase activation and apoptosis induction. Interestingly, in SHIP2-expressing breast cancer cells that lack SHIP1 expression, pan-SHIP1/2 inhibition also reduces viable cell numbers, which can be rescued by addition of exogenous PtdIns(3,4)P(2). In conclusion, this study shows that inhibition of SHIP1 and SHIP2 may have broad clinical application in the treatment of multiple tumor types.
Aside from being a phosphatase, SHIP1 also functions to mask receptor tails to prevent recruitment of other signaling proteins, or as an adaptor protein for proteins such as Shc, DOK1 and Grb2, and as such has been proposed to reduce Ras signaling. Theoretically, it is possible that while blocking phosphatase activity with 3AC, these other functions of SHIP1 may not be affected. However, we observed a decrease in SHIP1 protein expression in MM cells upon prolonged treatment with 3AC, suggesting that these scaffolding functions may no longer play a role. It has recently been shown that SHIP-1 is ubiquitinated and targeted for proteasomal degradation upon its phosphorylation. However, we did not observe a difference in IGF-1–stimulated SHIP1 phosphorylation in MM cells after pretreatment with 3AC (unpublished observations, GM Fuhler). Hence, the reason for the proteasomal degradation of SHIP1 upon 3AC treatment remains unclear.[2]

C27H49N

387.69

387.386

C, 83.65; H, 12.74; N, 3.61

2206-20-4

5351709

White to off-white solid powder

104.5-105.5℃ (methanol )

9.1

1

1

5

28

540

9

C[C@H](CCCC(C)C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC[C@@H]4[C@@]3(CC[C@H](C4)N)C)C

RJNGJYWAIUJHOJ-FBVYSKEZSA-N

InChI=1S/C27H49N/c1-18(2)7-6-8-19(3)23-11-12-24-22-10-9-20-17-21(28)13-15-26(20,4)25(22)14-16-27(23,24)5/h18-25H,6-17,28H2,1-5H3/t19-,20+,21-,22+,23-,24+,25+,26+,27-/m1/s1

(3R,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-amine

3α-Aminocholestane; 3AC; 3-AC; 2206-20-4; 3alpha-Aminocholestane; 3; A-Aminocholestane; (3alpha,5alpha)-Cholestan-3-amine; (3R,5S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-amine; 3??-Aminocholestane; 3+/--Aminocholestane; 3 AC

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3.25 mg/mL (8.38 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3.25 mg/mL (8.38 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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 (Please use freshly prepared in vivo formulations for optimal results.)