Tuspetinib Oral Myeloid Kinase Inhibitor Creates Synthetic Lethal

Vulnerability to Venetoclax

Himangshu Sonowal1, Ranjeet K. Sinha2, Rafael Bejar2, William Rice2, and Stephen Howell1

1UC San Diego Health, La Jolla, CA, USA; 2Aptose Biosciences Inc, San Diego, CA, USA

Background

Stability of Tuspetinib-Resistant Phenotype

Tuspetinib (HM43239; TUS), a once daily oral kinase inhibitor, suppresses key oncogenic signaling pathways operative in acute myeloid leukemia (AML) by inhibiting SYK, FLT3, RSK2, JAK1/2, mutant KIT, and TAK1-TAB1 kinases. In AML animal models, tuspetinib exhibited greater potency than gilteritinib and entospletinib when given as single agents and combined favorably with venetoclax (VEN) and azacytidine (AZA). Tuspetinib is being evaluated as monotherapy (TUS) and in combination (TUS/VEN) in a global Phase 1/2 trial of patients with R/R AML (NCT03850574). In AML, drug combination treatment overcomes drug-induced resistance. In this study, Tuspetinib resistant AML cells (TUS-Res) were created to investigate the resistance mechanism and sensitivity to other drugs.

Dissociation and inhibition constants for TUS

against key kinases operative in AML

Assay

Kinase

Mutation

Activity

Methodology

Status

WT

0.58

ITD

0.37

Binding Affinity

FLT3

D835Y

0.29

(KD, nM)

D835H

0.4

ITD/D835V

0.48

ITD/F691L

1.3

WT

1.1

FLT3

ITD

1.8

D835Y

1.0

SYK

WT

2.9

Inhibition of

JAK-1

2.8

JAK

JAK-2

6.3

Kinase Enzyme

Activity

JAK-2 (V617F)

9.9

(IC50, nM)

WT

> 500

c-KIT

D816H

3.6

D816V

3.5

RSK

RSK2

9.7

TAK1-TAB1

TAK1-TAB1

7.0

  • Stability of resistance to Tuspetinib was tested by removal Tuspetinib from TUS-Res cells and then monitoring for sensitivity to Tuspetinib for 15-,30-, and 60-days.TUS-Res clones maintained similar IC50 values for Tuspetinib, Gilteritinib and Venetoclax even

after removal of 75nM Tuspetinib for 60 days.

IC50 over 60 days following removal of Tuspetinib from growth medium of TUS-Res clones

TUS-Res Clone 3 TUS-Res Clone 4

50

200

50

200

05

10

8

ICuspetinibT

150

ICritinibGilte

150

ICVenetoclax

6

100

100

4

50

50

2

0

0

0

0

20

40

60

80

0

20

40

60

80

0

20

40

60

80

Days in TUS-free media

Days in TUS-free media

Days in TUS-free media

Apoptotic Machinery in Tuspetinib-Resistant Clones

•

In TUS-Res cells, the anti-apoptotic protein, Bcl-2 was marginally reduced and remained

stable over 30 days in the absence of Tuspetinib, whereas Mcl-1 expression was

unchanged. In contrast, parental cells showed higher Bcl-2 expression and reduction in

Mcl-1 expression after 48 hr treatment of high dose Tuspetinib.

•

The pro-apoptotic protein BAX was highly expressed in TUS-Res cells and retained stable

Objectives

  • Generate Tuspetinib-resistant(TUS-Res) AML cell lines
  • Investigate mechanistic changes in TUS-Res cell lines relative to parental
  • Investigate the vulnerabilities to other inhibitors of AML on TUS-Res cells

Methods

  • Generation of TUS-Rescells: MOLM-14 cells were grown in progressively higher concentrations of tuspetinib (TUS) over a period of 4 months. TUS-Res clones were then maintained in the presence of 75nM Tuspetinib and sub-cultured at 2-3 days.
  • Cytotoxicity assay: Tuspetinib was washed from TUS-Res cells to test the sensitivity of multiple inhibitors, including Gilteritinib, Quizartinib, Ruxolitinib, Fostamatinib, Venetoclax, Brequinar, Luxeptinib, IMP1088, 5-Azacytidine and Zelavespib. Cytotoxicity assays using CCK8 reagent were performed over a 72-h period of drug exposure, and inhibition of growth rate was determined.
  • Western Blot: Cell signaling targets and pro-andanti-apoptotic targets were analyzed by Western Blot. TUS-ResMOLM-14 cells (maintained in 75nM TUS) were analyzed for stability of phenotype by culturing cells in Tuspetinib-free media for 60 days (Sampling performed at 15, 30, and 60 days).
  • Statistical Analysis: Data reflect the mean ± SEM from 3 independent experiments and Student's t- test analysis. IC50was calculated using a curve fitting function in Prism.

Sensitivity of Tuspetinib-Resistant Cell Clones

  • Four clones of TUS-Res cells selected by continuous treatment of MOLM-14 cells in progressively higher concentrations of Tuspetinib for a period of 4 month
  • TUS-Resclones (maintained in 75nM Tuspetinib) were 139 ± 17-fold resistant to Tuspetinib
  • TUS-Resclones showed 2645-fold increased sensitivity to venetoclax
  • TUS-Resclones showed 156 ±22-fold resistance to gilteritinib
  • TUS-Resclones showed slightly increased sensitivity to quizartinib and luxeptinib (not shown)

expression for 30 days in the absence of Tuspetinib.

Western blot and quantification of apoptotic regulators in TUS-Res cells

BCL-2

MCL-1

BAX

Caspase-3

SYK-JAK-STAT5 signaling pathway is activated in

Tuspetinib-Resistant Clones

  • Relative to parental cells, TUS-Res cells showed an increase in phosphorylation of FLT3, JAK2, STAT5 and SYK, which persisted over 30 days after removal of Tuspetinib.
  • TUS-Rescells showed significantly higher levels of total FLT3 and STAT5 which indicates FLT3-mutant and Stat-5 survival pathways selection (Data not shown).

Effect of Tuspetinib on MOLM-14 parental and

Effect of Venetoclax on MOLM-14 parental

TUS-resistant clones

and TUS-resistant clones

150

MOLM-14 Parental

Growth rate (% Control)

150

MOLM-14 Parental

TUS-Res Clone 1

Growth rate (% Control)

TUS-Res Clone 3

TUS-Res Clone 2

TUS-Res Clone 4

100

100

TUS-Res Clone 3

TUS-Res Clone 4

50

50

0

0

1

1

1

1

0

00

000

0

0

00

0 .

1

0

0

0.1

1

10

100

000 10000

.

0

1

0

0

.

0

1

0

00

0

1

Log [Tuspetinib] (nM)

-50

1

Log [Venetoclax] (nM)

Phospho- FLT3

8

TUS-free

pFLT3/FLT3

(30 days)

6

***

4

***

*

2

0

pY591-FLT3

FLT3

GAPDH

Parental Clone 3 Clone 4

Clone 3 Clone 4

Phospho-SYK

SYK/SYK

20

TUS-free

(30 days)

15

*

***

pY525/526-

***

10

5

*

0

pSyk

Syk

Parental Clone 3 Clone 4

Clone 3 Clone 4

/JAK2

50

30

pJAK2

40

20

10

0

p-JAK2 JAK2

Phospho-JAK2

TUS-free (30 days)

**

* ** *

Parental Clone 3 Clone 4

Clone 3

Clone 4

Phospho-STAT5

STAT5/

3

***

TUS-free

***

2

(30 days)

-Y694STAT5

1

0

p-STAT5 STAT5

GAPDH

Parental Clone 3 Clone 4

Clone 3 Clone 4

-50

Effect of Gilteritinib on MOLM-14 parental and

Effect of Quizartinib on MOLM-14 parental and

TUS-resistant clones

TUS-resistant clones

Control)

150

MOLM-14 Parental

Control)

150

TUS-Res Clone 1

MOLM-14 Parental

100

TUS-Res Clone 2

100

TUS-Res Clone 1

TUS-Res Clone 3

TUS-Res Clone 2

(%

TUS-Res Clone 4

(%

TUS-Res Clone 3

50

TUS-Res Clone 4

rate

50

rate

Growth

0

0.1

1

10

100

100010000

Growth

0

0.1

1

00 10000

Log [Gilteritinib]

Log [Quizartinib] (nM)

-50

-50

IC50 (nM, Mean ± SD) of Various Drugs for

Tuspetinib-Resistant Clones

MOLM-14 parental

TUS-Res. Clone 1

TUS-Res. Clone 2

TUS-Res. Clone 3

TUS-Res. Clone 4

Tuspetinib

2.33 ± 0.28

183.18 ± 32.76

119.92 ± 13.88

107.96

± 11.32

144.40

± 14.86

Venetoclax

3016.37

± 172.91

ND

ND

1.14 ± 0.09

4.95 ± 0.38

Gilteritinib

8.58 ± 0.08

ND

ND

118.43 ± 3.99

128.07

± 4.20

Quizartinib

0.46 ± 0.01

ND

ND

0.21 ± 0.03

0.50 ± 0.04

Brequinar

116.20 ± 1.82

ND

ND

73.67

± 3.26

66.28

± 2.07

Luxeptinib

0.19 ± 0.01

ND

ND

0.06 ± 0.01

0.08 ± 0.003

IMP-1088

161.61

± 17.53

ND

ND

66.30

± 7.08

53.03

± 6.53

5-azacytidine

2137.67

± 109.00

ND

ND

1615.33

± 138.85

1580.67

± 68.47

Zelavespib

74.98

± 3.86

ND

ND

86.23

± 2.30

114.07

± 2.36

Fostamatinib

99.55. ± 10.25

ND

ND

175.65

± 10.95

459.25

± 28.72

Ruxolitinib

≈10000

ND

ND

≈10000

≈10000

Tuspetinib Targets Venetoclax-Resistance Mechanisms

  • TUS inhibits kinase-driven abnormal signaling
    • TUS reduces MCL-1 protein expression
    • VEN continues to inhibit BCL-2 block on cell death
    • TUS/VEN combine to kill AML cells
    • TUS can deliver responses in TP53MUT patients

Conclusions

  • Stable MOLM-14Tuspetinib-resistant clones (TUS-Res) were generated
  • TUS-Resclones showed 139 ±17-fold resistance to tuspetinib
  • TUS-Resclones remarkably are >2000-fold more sensitive to venetoclax
  • TUS-Resclones retain sensitivity to luxeptinib, quizartinib, IMP-1088,5-azacytidine, zelavespib, fostamatinib, and ruxolitinib similar to that of the parental MOLM-14 clone
  • TUS-Resclones have impaired apoptotic regulators
    ▪ Bcl-2 reduced │ Caspase 3, BAX, and BIM upregulated │ No change in Mcl-1 or PARP
  • TUS-Resclones expressed higher levels of phospho-FLT3,-STAT5,-SYK and -JAK2
  • TUS-Rescells are hypersensitive to Venetoclax, suggesting the TUS/VEN combination may prevent the development of resistance to Tuspetinib
  • Tuspetinib targets the resistance mechanisms of Venetoclax and the TUS/VEN combination may re-sensitizeVEN-resistant AML to Venetoclax - See the ESH2023 Abstract# 6046059

Disclosures: Current study is sponsored by Aptose Biosciences. The following authors are

employees of Aptose Biosciences: R Sinha, W Rice, and R Bejar. Contact: showell@health.ucsd.edu; rbejar@aptose.com

ND- Not Determined

ESH # 6044183

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Aptose Biosciences Inc. published this content on 29 October 2023 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 29 October 2023 10:26:41 UTC.