Methods for assessing and treating tyrosine kinase inhibitor (TKI)-resistant acute myeloid leukemia

Abstract

Disclosed herein is a method for assessing resistance to tyrosine kinase inhibitor (TKI) therapy of acute myeloid leukemia (AML) in a subject, comprising detecting the status of CD33, CD44, and phosphorylated BCL2-associated cell death agonist (pBAD) expression. Also disclosed herein is a method for treating AML in a subject by administering to the subject one or more inhibitors that target a TKI and a TKI-activated compensatory pathway.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 265,889, filed December 22, 2021, the entire contents of which are incorporated herein by reference. The present disclosure relates to compositions and methods for the treatment of cancer utilizing a combination of a therapeutic agent and an inhibitor that targets a treatment-activated compensation system. [Background technology]

[0002] Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy with the poorest survival rate among leukemias. Disease relapse is the most important cause of treatment failure. The standard treatment for AML is chemotherapy with a combination of anthracyclines and cytarabine. In addition, targeted therapies for AML based on the identification of gene mutations using next-generation sequencing have been developed. FLT3-like receptor tyrosine kinase 3 (FLT3) is expressed in many AMLs, and 35% of AML patients have oncogenic FLT3 mutations, such as internal tandem duplications (ITDs) or point mutations in the TK domain (TKD) of this protein. FLT3 mutations are associated with reduced overall survival with an increased risk of relapse in AML. Mechanistically, constitutive activation of mutant FLT3 stimulates multiple downstream signaling pathways, including PI3K-ACT, RAS-RAF-ERK, and JAK / STAT5, promoting cell survival and proliferation.

[0003] Several tyrosine kinase inhibitors (TKIs), including midostaurin (MIDO), sorafenib (SORA), gilteritinib (GILT), and quizartinib (QUIZ), have been evaluated in clinical trials for the treatment of AML with FLT3 mutations, some of which have been approved by the U.S. Food and Drug Administration (FDA). However, the anti-leukemic efficacy of the recently approved FLT3 inhibitors as monotherapy or in combination with standard treatment is limited by short duration and high relapse rates. Patients with relapsed or refractory AML with mutations in FLT3 have poor response to salvage chemotherapy. Even with new generation FLT3 inhibitors, the median overall survival is less than 1 year. Although epigenetic and proteomic profiles of AML have been reported, specific immunophenotypic biomarkers of TKI-resistant blasts have yet to be identified. Summary of the Invention

[0004] Provided herein are systems and methods that address these shortcomings in the art, including developing effective therapies to address AML relapse after TKI treatment and overcome TKI resistance, and provide other additional or alternative advantages. Disclosed herein are methods of assessing resistance of AML in a subject to TKI therapy. One such method includes isolating AML tumor cells from a subject and detecting the status of markers CD33, CD44, and phosphorylated BCL2-associated cell death agonist (pBAD) on the surface of the AML tumor cells, determining whether the tumor cells are CD33 positive (CD33 + ), CD44 high positive (CD44high + ), and pBAD positive (pBAD + In some embodiments, the subject is determined to be at high risk for developing TKI-resistant AML in response to CD33 + CD44high + pBAD + The tumor cells were also positive for the nuclear protein Ki67 (Ki67 +). Also disclosed herein are embodiments of methods of treating acute myeloid leukemia (AML) in a subject. One such method includes administering to the subject a therapeutically effective amount of a TKI and a therapeutically effective amount of an inhibitor of a TKI-activated compensatory pathway or an inhibitor of intracellular homeostasis required for leukemia relapse after treatment. In some embodiments, the inhibitor inhibits the JAK-STAT pathway, the TYK2-STAT4-PIM2 / PIM3 pathway, and / or the NFκB2 (P100 / P52)-MIF-CXCR2 pathway. In some embodiments, the inhibitor is one or more of a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a TKI, a therapeutically effective amount of a BCL2 inhibitor, and a therapeutically effective amount of an NFκB inhibitor. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a TKI, a therapeutically effective amount of a STAT4 inhibitor ... TKI is GILT or QUIZ. In some embodiments, the STAT4 inhibitor is (R)-lisofylline. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the NFκB inhibitor is MG-132 or BAY11-7082. In some embodiments, the CD44 inhibitor is Angstrom6, a urokinase-derived peptide. In some embodiments, the CXCR2 inhibitor is SB225002. In some embodiments, the subject has TKI-resistant AML or is at risk of developing TKI-resistant AML.

[0005] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments will be readily understood from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. [Brief description of the drawings]

[0006] [Figure 1A]Figure 1 shows the therapeutic effect of different TKIs on MV4-11 and MOLM-14 cells in vitro (N=6). Figure 1A shows representative FACS (fluorescence-activated single-cell sorting) plots for MV4-11 cells after 3 days of in vitro treatment with different TKI inhibitors and 5-azacytidine (AZA) and 1α,25-dihydroxyvitamin D3 (VD3). [Figure 1B] Figure 1B shows the therapeutic effect of different TKIs on MV4-11 and MOLM-14 cells in vitro (N=6). Figure 1B shows representative FACS plots for MOLM-14 cells after 3 days of in vitro treatment with different TKI inhibitors and AZA / VD3. The circles indicate the groups of viable blasts located on the FACS plot, and the green arrows indicate the tendency of blasts to die, becoming viability dye positive over the next few days. [Figure 1C] Figure 1 shows the therapeutic effect of different TKIs on MV4-11 and MOLM-14 cells in vitro (N=6). Figures 1C and 1D are graphical representations of cumulative FACS percentage data of viable MV4-11 and MOLM-14 cells. *P<0.05. [Figure 1D] Figure 1 shows the therapeutic effect of different TKIs on MV4-11 and MOLM-14 cells in vitro (N=6). Figures 1C and 1D are graphical representations of cumulative FACS percentage data of viable MV4-11 and MOLM-14 cells. *P<0.05. [Figure 1E] Figure 1 shows the therapeutic effect of different TKIs on MV4-11 and MOLM-14 cells in vitro (N=6). Figure 1E shows a representative FACS plot of Ki67 expression in different treatment groups after 3 days of in vitro treatment. The black arrows indicate the Ki67 expression curves and indicate treatment groups with lower Ki67 expression compared to untreated controls. [Figure 2A]Figure 2 shows that TKI-treated blasts underwent tumorigenic clustering regrowth after 10 days of in vitro treatment with different TKIs (N=6). Figure 2A shows representative phase contrast images showing differential regrowth of TKI-treated MV4-11 and MOLM-14 cells. The arrows in the images of GILT-treated cells indicate many repopulated tumorigenic clusters in GILT-treated blasts. The arrows in the images of QUIZ-treated cells indicate few repopulated tumorigenic clusters in QUIZ-treated blasts. Scale bar: 100 μm. In in vitro studies of TKI-treated MV4-11 and TKI-treated MOLM-14, the patterns of blast regrowth, including timing and immunophenotype, are similar. [Figure 2B] Figure 2B shows how TKI-treated blasts underwent tumorigenic clustering regrowth after 10 days of in vitro treatment with different TKIs (N=6). Figure 2B shows representative FACS plots of pBAD expression in MIDO and SORA experimental groups after 3 days of in vitro treatment. The expression curves of pBAD in the different experimental groups show that the treatment groups have more pBAD expression in both MV4-11 and MOLM-14 cells when compared to the untreated control. [Figure 2C] Figure 2 shows that TKI-treated blasts underwent tumorigenic clustering regrowth after 10 days of in vitro treatment with different TKIs (N=6). Figure 2C is a set of representative FACS plots of CD44+pBAD+ populations in MV4-11 cells treated with different TKI inhibitors after 10 days of continuous in vitro culture. Arrows indicate CD44+pBAD+ cells in GILT-treated and QUIZ-treated blasts. Boxes indicate high expression of CD44 ("CD44high+") in GILT-treated and QUIZ-treated blasts. [Figure 2D] Figure 2 shows that TKI-treated blasts underwent tumorigenic clustering regrowth after 10 days of in vitro treatment with different TKIs (N=6). Figure 2D is a graphical representation of cumulative FACS percentage data of viable CD44+pBAD+MV4-11 cells. *P<0.05. [Figure 2E]Figure 2 shows that TKI-treated blasts underwent tumorigenic clustering regrowth after 10 days of in vitro treatment with different TKIs (N=6). Figure 2E is a set of representative FACS plots of Ki67+CD33+CD44+ populations in MV4-11 cells treated with different TKIs after continuous in vitro culture. Arrows indicate Ki67+CD33+CD44+ cells in GILT-treated and QUIZ-treated blasts. Circles indicate Ki67+CD33+CD44+ cell populations in the experimental groups of control, MIDO-treated and SORA-treated MV4-11 cells. [Figure 2F] Figure 2 shows that TKI-treated blasts underwent tumorigenic clustering regrowth after 10 days of in vitro treatment with different TKIs (N=6). Figure 2F is a graphical representation of FACS percentage data of viable Ki67+CD33+CD44+MV4-11 cells. *P<0.05. [Figure 3A] GILT-treated MV4-11 cells recover and grow confluently after 20 days of in vitro treatment (N=6). Figure 3A is a set of representative phase contrast bright images showing control and TKI-treated MV4-11 cells. Arrows indicate tumorigenic clusters in QUIZ-treated blasts. Scale bar: 50 μm. [Figure 3B] Figure 3 shows that GILT-treated MV4-11 cells recover and grow confluently after 20 days of in vitro treatment (N=6). Figure 3B shows a set of representative FACS plots of CD44+pBAD+ populations in MV4-11 cells treated with different TKIs after 20 days of continuous in vitro culture. Arrows indicate CD44+pBAD+ cells in GILT-treated and QUIZ-treated blasts. [Figure 3C] GILT-treated MV4-11 cells recover and grow confluently after 20 days of in vitro treatment (N=6). FIG. 3C is a graphical representation of cumulative FACS percentage data of viable CD44+pBAD+MV4-11 cells. Arrows indicate GILT-treated and QUIZ-treated blasts. **P<0.01. [Figure 3D]GILT-treated MV4-11 cells recover and grow confluently after 20 days of in vitro treatment (N=6). FIG. 3D is a set of representative FACS plots of Ki67+CD33+CD44+ populations in MV4-11 cells treated with different TKIs after 20 days of continuous in vitro culture. Arrows indicate Ki67+CD33+CD44+ cells in GILT-treated and QUIZ-treated blasts. **P<0.01. [Figure 3E] GILT-treated MV4-11 cells recover and grow confluently after 20 days of in vitro treatment (N=6). FIG. 3E is a graphical representation of cumulative FACS percentage data of viable Ki67+CD33+CD44+MV4-11 cells. Arrows indicate GILT-treated and QUIZ-treated blasts. **P<0.01. [Figure 4A] Figure 4. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (3, 10, 20 days) analyzed by qPCR. After 3, 10, and 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 4A is a set of graphical representations of mRNA expression of genes required for cell death pathways 3 days after treatment. In Figures 4A-4E, data are means ± SEM where applicable. *P<0.05, **P<0.01, N=4-6. [Figure 4B] Figure 4. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (days 3, 10, and 20) analyzed by qPCR. After 3, 10, and 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4B and 4C are a set of graphical representations of mRNA expression of genes required for phosphorylation of BAD protein 3 days after treatment. In Figures 4A-4E, data are means ± SEM, where applicable. *P<0.05, **P<0.01, N=4-6. [Figure 4C]Figure 4. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (days 3, 10, and 20) analyzed by qPCR. After 3, 10, and 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4B and 4C are a set of graphical representations of mRNA expression of genes required for phosphorylation of BAD protein 3 days after treatment. In Figures 4A-4E, data are means ± SEM, where applicable. *P<0.05, **P<0.01, N=4-6. [Figure 4D] Figure 4. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (3, 10, 20 days) analyzed by qPCR. After 3, 10, and 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 4D is a set of graphical representations of mRNA expression of genes required for the JAK-STAT signaling pathway 3 days after treatment. In Figures 4A-4E, data are means ± SEM, where applicable. *P<0.05, **P<0.01, N=4-6. [Figure 4E] Figure 4. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (days 3, 10, and 20) analyzed by qPCR. After 3, 10, and 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 4E is a set of graphical representations of mRNA expression of genes required for intracellular inhibitory pathways 3 days after treatment. In Figures 4A-4E, data are means ± SEM, where applicable. *P<0.05, **P<0.01, N=4-6. [Figure 4F]Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4F-4I are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figures 4F and 4H) and for reprogramming of intracellular inhibitory pathways (Figures 4G and 4I) 10 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4G] Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4F-4I are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figures 4F and 4H) and for reprogramming of intracellular inhibitory pathways (Figures 4G and 4I) 10 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4H] Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4F-4I are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figures 4F and 4H) and for reprogramming of intracellular inhibitory pathways (Figures 4G and 4I) 10 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4I]Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4F-4I are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figures 4F and 4H) and for reprogramming of intracellular inhibitory pathways (Figures 4G and 4I) 10 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4J] Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4J-4M are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figure 4J and Figure 4L) and for reprogramming of intracellular inhibitory pathways (Figure 4K and Figure 4M) at 20 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4K] Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4J-4M are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figure 4J and Figure 4L) and for reprogramming of intracellular inhibitory pathways (Figure 4K and Figure 4M) at 20 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4L]Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4J-4M are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figure 4J and Figure 4L) and for reprogramming of intracellular inhibitory pathways (Figure 4K and Figure 4M) at 20 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 4M] Figure 4. Pro-survival gene expression changes in MV4-11 cells treated with TKIs in vitro analyzed by qPCR (3, 10, 20 days). After 3, 10, 20 days of in vitro treatment with different TKIs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figures 4J-4M are a set of graphical representations of mRNA expression of genes required for compensatory pathways (Figure 4J and Figure 4L) and for reprogramming of intracellular inhibitory pathways (Figure 4K and Figure 4M) at 20 days after in vitro treatment with different TKI drugs. In Figures 4F-4M, data are means ± SEM, where applicable. *P<0.05, N=4. [Figure 5A] QUIZ-treated MV4-11 recovered and grew confluently after 28 days in vitro (N=6). Figure 5A is a set of representative FACS plots of CD44+pBAD+ populations in MV4-11 cells treated with different TKIs after continuous in vitro culture. Arrows indicate CD44+pBAD+ cells in QUIZ-treated blasts. [Figure 5B] Figure 5B shows that QUIZ-treated MV4-11 recovered and grew confluently after 28 days in vitro (N=6). Figure 5B is a set of representative FACS plots of Ki67+CD33+CD44+ populations in MV4-11 cells treated with different TKI inhibitors after 28 days of continuous culture in vitro. Arrows indicate Ki67+CD33+CD44+ cells in QUIZ-treated blasts. [Figure 5C] QUIZ-treated MV4-11 recovered and grew to confluence after 28 days in vitro (N=6). Figures 5C and 5D are graphical representations of cumulative FACS percentage data of viable CD44+pBAD+MV4-11 cells and Ki67+CD33+CD44+MV4-11 cells. *P<0.05. [Figure 5D] QUIZ-treated MV4-11 recovered and grew to confluence after 28 days in vitro (N=6). Figures 5C and 5D are graphical representations of cumulative FACS percentage data of viable CD44+pBAD+MV4-11 cells and Ki67+CD33+CD44+MV4-11 cells. *P<0.05. [Figure 6A] Figure 6. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (28 days) analyzed by qPCR. After 28 days of in vitro treatment with different TKI drugs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 6A is a set of graphical representations of mRNA expression of genes required for cell death pathways. Data are means ± SEM where applicable. *P<0.05, N=4-6. [Figure 6B] Figure 6A shows changes in pro-survival gene expression in MV4-11 following in vitro TKI treatment (28 days) analyzed by qPCR. After 28 days of in vitro treatment with different TKI drugs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 6B is a set of graphical representations of mRNA expression of genes required for phosphorylation of BAD protein. Data are means ± SEM where applicable. *P<0.05, N=4-6. [Figure 6C]Figure 6A shows changes in pro-survival gene expression in MV4-11 following in vitro TKI treatment (28 days) analyzed by qPCR. After 28 days of in vitro treatment with different TKI drugs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 6C is a set of graphical representations of mRNA expression of genes required for the JAK-STAT signaling pathway. Data are means ± SEM where applicable. *P<0.05, N=4-6. [Figure 6D] Figure 6. Pro-survival gene expression changes in MV4-11 following in vitro TKI treatment (28 days) analyzed by qPCR. After 28 days of in vitro treatment with different TKI drugs, cells were harvested and RNA isolation was performed as described in Materials and Methods. Gene expression was analyzed by qPCR. Figure 6D is a set of graphical representations of mRNA expression of genes required for intracellular inhibitory pathways. Data are means ± SEM where applicable. *P<0.05, N=4-6. [Figure 7A] Macrophage migration inhibitory factor (MIF) release from AML blasts. Figure 7A is a set of images from a partial blot film made for proteomic analysis of cell-free supernatant from MV4-11 cells treated with 80 nM GILT. The dotted arrow indicates osteopontin at the same position in the film. The shaded arrow indicates MIF dot at the same position in the film. Each antibody has two dot spots according to the manufacturer's specifications. [Figure 7B] Figure 7 shows the release of macrophage migration inhibitory factor (MIF) from AML blasts. Figure 7B is a graphical representation of the cumulative mean pixel density of MIF (fold change). **P<0.01. [Figure 7C] Figure 7C shows the release of macrophage migration inhibitory factor (MIF) from AML blasts. Figure 7C is a set of images of a partial blot film made for proteomic analysis of cell-free supernatant from primary AML BMMNC cells (patient no. 1) treated with 80 nM GILT. The solid arrow indicates the reference spot (control dot) from the manufacturer. The shaded arrow indicates the dot of MIF at the same position in the film. [Figure 7D] Figure 7D shows the release of macrophage migration inhibitory factor (MIF) from AML blasts. Graphical representation of the cumulative mean pixel density of MIF (fold change). **P<0.01. [Figure 7E] Figure 7E shows the release of macrophage migration inhibitory factor (MIF) from AML blasts. Figure 7E is a set of graphical representations of qPCR gene expression analysis 3 days after treatment with different TKIs in vitro. Data show the mRNA expression of genes encoding the different receptors for MIF, CD74, CD44, CXCR4 and CXCR2. *P<0.05, **P<0.01. (MIF) in MV4-11. [Figure 8A] Figure 8 shows that MIF promotes MV4-11 proliferation by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8A shows a representative FACS histogram plot of Ki67 expression in MV4-11 experimental groups after 2 days in vitro. The treated groups showed higher expression of Ki67 compared to the untreated control group. [Figure 8B] Figure 8 shows that MIF promotes MV4-11 proliferation by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8B shows a representative FACS histogram plot of Ki67 expression in RAW264.7 experimental groups after 2 days in vitro. The treated groups showed less Ki67 expression compared to the untreated control group. MIF promoted the survival of CD44High+ cells after TKI treatment in vitro. [Figure 8C] Figure 8C shows that MIF promotes MV4-11 proliferation by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8C is a set of representative FC plots of Ki-67 and CD44 expression in MV4-11 experimental groups after sequential co-culture with 80 nM GILT and appropriate doses of MIF for 5 days in vitro. Arrows indicate viable Ki-67-CD44+ cells. Figure 8C also includes a graphical representation of the cumulative FC percentage data of viable Ki-67-CD44+ cells on the right side. *p<0.05. [Figure 8D] Figure 8D shows that MIF promotes MV4-11 proliferation by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8D is a set of representative FC plots of Ki-67 and CD44 expression in MV4-11 experimental groups after 5 days of co-culture with 80 nM GILT and appropriate doses of MIF in vitro. Arrows indicate Ki-67+CD44+ or Ki-67-CD44+ cell populations, respectively. Figure 8D also includes a graphical representation of the cumulative FC percentage data of viable CD44High+ cells on the right side. *p<0.05. [Figure 8E] Figure 8E shows that MIF promotes MV4-11 proliferation by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8E is a representative FC histogram plot of CD44 expression in MV4-11 experimental groups after 5 days of in vitro co-culture. Each group was treated with 80 nM GILT alone or in combination with different doses of MIF. The GILT-treated group supplemented with MIF had a CD44High+ cell population compared to the untreated control or GILT alone. Figure 8E also includes on the right a graphical representation of the change in mRNA expression of CXCR2 gene in MV4-11 cells in different doses of MIF combined with 80 nM GILT in vitro. *p<0.05, **p<0.01. [Figure 8F] Figure 8 shows that MIF promotes MV4-11 proliferation by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8F is a set of graphical representations of the changes in mRNA expression of CXCL1, 5, 8 chemokine genes in MV4-11 cells in vitro with different doses of MIF. *P<0.05, **P<0.01. [Figure 8G] Figure 8G shows that MIF promotes the proliferation of MV4-11 by upregulating the expression of CXCR2, cytokines, and cell division proteins. Figure 8G is a set of graphical representations of the changes in mRNA expression of CDK4 and CYCLIN E1 genes in MV4-11 cells in different doses of MIF in vitro. *P<0.05. [Figure 9A] Figure 14. MIF and TKI treatment-induced CXCR2 expression in MV4-11 cells in vitro. *p<0.05, **p<0.01. [Figure 9B] Figure 14. MIF and TKI treatment-induced CXCR2 expression in MV4-11 cells in vitro. *p<0.05, **p<0.01. [Figure 9C] Figure 9 shows MIF and TKI treatment-induced CXCR2 expression in MV4-11 cells in vitro. Figure 9C shows NFκB2 activation induced by TKI treatment. *p<0.05, **p<0.01. [Figure 10] FIG. 1 is a set of representative FACS plots of viable MV4-11 cells (indicated by arrows) in different treatment groups, showing the therapeutic effect of combination therapy of 20 μg / ml RPS19 (MIF-I) and 80 nM GILT on MV4-11 after 3 days in vitro. [Figure 11A] Figure 11 shows the therapeutic effect of GILT and NFκB inhibitors on MV4-11 cells and primary AML-FLT3 BMMNC cells. Figure 11A shows a set of representative FACS plots of viable CD44+ MV4-11 cells in different treatment groups. The arrows indicate further analysis of Ki67 expression of these viable CD44+ blasts. [Figure 11B] Figure 11 shows the therapeutic effect of GILT and NFκB inhibitors on MV4-11 cells and primary AML-FLT3 BMMNC cells. Figure 11B is a graphical representation of cumulative FACS percentage data of viable CD44+ MV4-11 cells. [Figure 11C] Figure 11 shows the therapeutic effect of GILT and NFκB inhibitors on MV4-11 cells and primary AML-FLT3 BMMNC cells. Figure 11C is a graphical representation of cumulative FACS percentage data of viable Ki67+CD44+MV4-11 cells. [Figure 11D]Figure 11 shows the therapeutic effect of GILT and NFκB inhibitors on MV4-11 cells and primary AML-FLT3 BMMNC cells. Figure 11D shows a set of representative FACS plots of viable CD33+CD13+ primary blasts (patient number 3) in different treatment groups. The arrows indicate further analysis of Ki67 expression of these viable CD33+CD13+ primary blasts. [Figure 11E] Figure 11 shows the therapeutic effect of GILT and NFκB inhibitors on MV4-11 cells and primary AML-FLT3 BMMNC cells. Figure 11E is a graphical representation of the cumulative FACS percentage data of viable CD33+CD13+ primary blasts. [Figure 11F] FIG. 1 shows the therapeutic effects of GILT and NFκB inhibitors on MV4-11 cells and primary AML-FLT3 BMMNC cells. [Figure 12A] Figure 12 shows the therapeutic effect of NFκB inhibitors and siRNA knockdown of NFκB2 in bone marrow mononuclear cells (BMMNC). Figure 12A is a set of graphical representations of the mRNA expression of the canonical pathway (NFκB1) and non-canonical pathway (NFκB2) after different TKI treatments (80 nM). [Figure 12B] Figure 12 shows the therapeutic effect of NFκB inhibitors and siRNA knockdown of NFκB2 in bone marrow mononuclear cells (BMMNC). Figure 12B is a graphical representation of NFκB2 mRNA expression after 2 days of siRNA-NFκB2 treatment (50 nM) with or without 80 nM GILT in vitro. Cells were harvested for RNA isolation and gene expression was analyzed by qPCR (N=3). [Figure 12C] Figure 12 shows the therapeutic effect of NFκB inhibitors and siRNA knockdown of NFκB2 in bone marrow mononuclear cells (BMMNC). Figures 12C, 12D and 12E show the graphical representation of the mRNA expression of MIF, CXCR2 and CXCL5 genes, respectively, after transient knockdown of NFκB2 in MV4-11 cells. *P<0.05, **P<0.01. [Figure 12D]Figure 12 shows the therapeutic effect of NFκB inhibitors and siRNA knockdown of NFκB2 in bone marrow mononuclear cells (BMMNC). Figures 12C, 12D and 12E show the graphical representation of the mRNA expression of MIF, CXCR2 and CXCL5 genes, respectively, after transient knockdown of NFκB2 in MV4-11 cells. *P<0.05, **P<0.01. [Figure 12E] Figure 12 shows the therapeutic effect of NFκB inhibitors and siRNA knockdown of NFκB2 in bone marrow mononuclear cells (BMMNC). Figures 12C, 12D and 12E show the graphical representation of the mRNA expression of MIF, CXCR2 and CXCL5 genes, respectively, after transient knockdown of NFκB2 in MV4-11 cells. *P<0.05, **P<0.01. [Figure 13A] Figure 13 shows the therapeutic effect of a combination of 50μM NFκB inhibitor and 80nM GILT in newly diagnosed AML-FLT3 BMMNC (patient number 5). Figure 13A is a set of representative FACS plots of viable CD33+CD13+ primary blasts in different treatment groups. The arrows indicate further analysis of Ki67 expression of these viable CD33+CD13+ primary blasts. [Figure 13B] Figure 13B shows the therapeutic effect of a combination of 50 μM NFκB inhibitor and 80 nM GILT in newly diagnosed AML-FLT3 BMMNC (patient number 5). Figure 13B is a graphical representation of the cumulative FACS percentage data of viable CD33+CD13+ primary blasts. [Figure 13C] Figure 13C shows the therapeutic effect of a combination of 50 μM NFκB inhibitor and 80 nM GILT in newly diagnosed AML-FLT3 BMMNC (patient number 5). Figure 13C is a graphical representation of the cumulative FACS percentage data of viable Ki67+CD33+CD13+ primary blasts. [Figure 13D]Figure 13D shows the therapeutic effect of a combination of 50 μM NFκB inhibitor and 80 nM GILT in newly diagnosed AML-FLT3 BMMNC (patient no. 5). Figure 13D is a set of images of a partial blot film made for proteomic analysis of cell-free supernatant from primary AML BMMNC cells treated with GILT. Solid arrows indicate control dots from the manufacturer. Diagonal, shaded and dotted arrows indicate dots of CXCL1, CXL5 and CXCL8 in the film, respectively. [Figure 13E] Figure 13E shows the therapeutic effect of a combination of 50 μM NFκB inhibitor and 80 nM GILT in newly diagnosed AML-FLT3 BMMNC (patient #5). Figure 13E is a set of graphical representations of cumulative mean pixel density (fold change) of CXCL1, CXCL5, CXCL8. *P<0.05, **P<0.01. [Figure 14A] Figure 14 shows the therapeutic effect of the combination of 50 μM NFκB inhibitor and 80 nM GILT in refractory AML-FLT3-ITDBMMNC cells (number 7). Figure 14A is a set of representative phase contrast bright images showing tumorigenic cluster formation in different experimental groups after 3 days of treatment with different TKI drugs ex vivo. [Figure 14B] Figure 14B shows the therapeutic effect of the combination of 50μM NFκB inhibitor and 80nM GILT in refractory AML-FLT3-ITDBMMNC cells (number 7). Figure 14B is a set of representative FACS plots of viable CD117+CD13+ primary blasts in different treatment groups. The arrows indicate further analysis of Ki67 expression of these viable CD117+CD13+ primary blasts. [Figure 14C] Figure 14 shows the therapeutic effect of the combination of 50 μM NFκB inhibitor and 80 nM GILT in refractory AML-FLT3-ITDBMMNC cells (number 7). Figure 14C and Figure 14D are graphical representations of the mRNA expression of CYCLIN E1 and CD44 genes, respectively, after 3 days of ex vivo treatment. Cells were harvested for RNA isolation and gene expression was analyzed by qPCR. *P<0.05, **P<0.01. [Figure 14D]Figure 14 shows the therapeutic effect of the combination of 50 μM NFκB inhibitor and 80 nM GILT in refractory AML-FLT3-ITDBMMNC cells (number 7). Figure 14C and Figure 14D are graphical representations of the mRNA expression of CYCLIN E1 and CD44 genes, respectively, after 3 days of ex vivo treatment. Cells were harvested for RNA isolation and gene expression was analyzed by qPCR. *P<0.05, **P<0.01. [Figure 15] Graphical representation of cumulative mean pixel density (fold change) of CXCL8 obtained from proteomic analysis of cell-free supernatants from treatment groups of primary AML BMMNC cells. *P<0.05, **P<0.01. [Figure 16A] Figure 16 shows the therapeutic effect of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figure 16A is a set of representative FACS plots of viable blast populations in different combination treatments on MV4-11 cells in vitro. Circles indicate viable blasts in the FACS plots. Arrows indicate the tendency of blasts to die, becoming viability dye positive over the next few days. [Figure 16B] Figure 16 shows the therapeutic effect of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figure 16B is a graphical representation of the cumulative FACS percentage data of viable MV4-11 cells in different experimental groups at 3 days after in vitro treatment. *P<0.05, **P<0.01. [Figure 16C] Figure 16 shows the therapeutic effect of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figure 16C shows a set of representative FACS plots of Ki67+CD33+CD44+ populations after continuous in vitro culture (7 days after treatment) for different combination therapies on MV4-11 cells. [Figure 16D]Figure 16 shows the therapeutic effect of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figure 16D shows a graphical representation of the cumulative cell counts of Ki67+CD33+CD44+MV4-11 in different experimental groups. *P<0.05, **P<0.01. [Figure 16E] Therapeutic effects of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figures 16E-16H are representative phase contrast images of MV4-11 cells with and without GILT-based combination treatment as indicated. Scale bar: 100 μm. Arrows indicate tumorigenic clusters in GILT-treated and GILT / STAT4 inhibitor-treated blasts. [Figure 16F] Therapeutic effects of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figures 16E-16H are representative phase contrast images of MV4-11 cells with and without GILT-based combination treatment as indicated. Scale bar: 100 μm. Arrows indicate tumorigenic clusters in GILT-treated and GILT / STAT4 inhibitor-treated blasts. [Figure 16G] Therapeutic effects of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figures 16E-16H are representative phase contrast images of MV4-11 cells with and without GILT-based combination treatment as indicated. Scale bar: 100 μm. Arrows indicate tumorigenic clusters in GILT-treated and GILT / STAT4 inhibitor-treated blasts. [Figure 16H] Therapeutic effects of GILT-based combination treatment on MV4-11 at 3 and 7 days after in vitro treatment (N=4). Figures 16E-16H are representative phase contrast images of MV4-11 cells with and without GILT-based combination treatment as indicated. Scale bar: 100 μm. Arrows indicate tumorigenic clusters in GILT-treated and GILT / STAT4 inhibitor-treated blasts. [Figure 17A]FIG. 1 is an illustration of the survival mechanism of CD33+CD44+pBAD+ blasts after TKI treatment. [Figure 17B] FIG. 1 is an illustration of intracellular changes in CD33+CD44+pBAD+ blast cells following TKI treatment. [Figure 17C] FIG. 1 is an illustration of TKI activation pathways involved in blast survival and proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Relapsed or refractory AML patients with mutations in the FLT3 gene are less responsive to salvage chemotherapy. Even with new generation FLT3 inhibitors, the median overall survival is less than one year. Resistance to such inhibitors, whether primary or secondary, inevitably occurs with the course of treatment, suggesting that alternative pathways are involved in the survival of relapsed / refractory leukemia cells. Evolutionarily, duplicated gene pairs ("redundant" elements) in cell systems may have redundant functions to ensure biological robustness for all organisms. However, the concept of "redundant gene" treatment-activated compensation systems is not well known in the AML field, and their potential role in AML relapse has not been explored. Thus, the present disclosure provides a method for detecting tyrosine kinase inhibitor (TKI)-resistant AML blasts by determining the levels of novel biomarkers CD33, CD44, phosphorylated BAD (pBAD), and combinations thereof, in subjects with or at risk of having AML. The present disclosure also provides combination therapy of a TKI with one or more inhibitors targeting the TKI-activated compensation pathway. The targeted TKI-activated compensatory pathways include the TYK2-STAT4-PIM2 / 3 pathway, which is a TKI-activated cell survival mechanism, and the NFκB2-MIF-CXCR2 pathway, which is a TKI-activated cell proliferation mechanism. Such combination therapy can treat AML more effectively than TKI monotherapy.

[0008] A "therapeutically effective amount" is an amount sufficient to produce a desired clinical result (i.e., achieve a therapeutic effect). A therapeutically effective dose may be administered in one or more administrations. "Administration" refers to the physical introduction of a therapeutic agent to a subject in need thereof. Exemplary routes of administration of agents for treating AML include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, including, for example, administration by injection or infusion. Modes of administration include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Therapeutic agents may be administered parenterally or orally. Other routes include, for example, topical, epidermal, or mucosal routes of administration, such as nasal, vaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time. The therapeutic agent can be made up into a composition, e.g., a pharmaceutical composition, which includes the agent and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible.

[0009] The present disclosure describes various embodiments of compositions and methods for the management or treatment of cancer, such as AML, gastric cancer, or breast cancer. In the following description, numerous details are set forth to provide a thorough understanding of the various embodiments. Before the methods and compositions are described, it should be understood that these embodiments are not limited to the specific methods or compositions described, as such may, of course, vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting, as the scope of the present embodiments is limited only by the appended claims. The phrases "in certain embodiments," "in various embodiments," "in one embodiment," or "in an embodiment" may be used herein, each of which may refer to one or more of the same or different embodiments. Furthermore, terms such as "comprising," "including," "having," and the like, used in connection with the embodiments of the present disclosure, are synonymous.

[0010] "Subject" refers to animals such as mammals, including primates (humans, non-human primates, such as monkeys) and non-primates (such as mice). In some aspects of the invention, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, infant, or child. In other aspects, the subject is an adult subject. "Patient" refers to a subject who exhibits symptoms and / or signs of a disease, is undergoing treatment for a disease, has been diagnosed with a disease, and / or is at risk for developing a disease. A "patient" can be a human or veterinary subject. Reference to a subject in this disclosure should be understood to include the possibility that the subject is a "patient" unless the context clearly dictates otherwise. More specifically, the subject in certain embodiments is a patient with a liquid cancer, such as leukemia.

[0011] As used herein, the terms "treat", "treatment" and the like include the management and care of a subject or patient for the purpose of combating a disease, condition, or disorder, and include the administration of a composition to prevent the onset of symptoms or complications, alleviate symptoms or complications, reduce at least one associated sign, symptom, or condition, or eliminate the disease, condition, or disorder. Treatment also refers to prophylactic treatment, such as the prevention of a disease (e.g., AML) or the prevention of at least one sign, symptom, or condition associated with a disease (e.g., recurrence of AML). Treatment can also mean the prolongation of survival compared to expected survival in the absence of treatment.

[0012] As used herein, the term "marker" refers to a marker that is a specific molecule that is a specific target of immunological testing. + " refers to the positive status or presence of said marker. As used herein, " " used in the context of a biomarker, or a cell surface marker. - " means the negative status or absence of said marker. The positive / negative status of a biomarker, cell surface marker, or amount of a cellular molecule can be determined by any method known in the art, including flow cytometry, Western blot, ELISA, and PCR. In some embodiments, the disclosed methods inhibit the TKI-activated TYK2-STAT2 / 4-PIM2 / 3 compensatory pathway. TKI-resistant blasts initially show a higher pBAD than non-TKI-resistant blasts. +TKI-resistant blasts show high CD44 expression along with β-blockade. The increased expression of CD44 and pBAD in resistant blasts gradually decreases as they regain their ability to survive, cluster, and grow confluently. Mechanistically, CD44 and its variant isoforms are known to activate different downstream signaling pathways, such as PI3K / ACT and Src / MAPK, leading to cancer cell invasion and proliferation. Blocking hyaluronan, the major ligand of CD44, inhibits phosphorylation of BAD. Furthermore, mRNAs of PIM2, PIM3, and AKT1 are significantly increased in TKI-resistant blasts. After TKI treatment, the increase in CD44 may result in the activation of the PIM2 / 3 kinase compensation pathway. Thus, the TKI-suppressed PIM1 kinase activity is restored to phosphorylate BAD protein and prevent its binding to BCL-2, allowing blasts to survive TKI treatment. In cellular systems, redundant factors may compensate for each other, such as activation of PIM2 to compensate for the lack of PIM1. In hepatocellular carcinoma, the JAK / STAT (STAT3) pathway is involved in the regulation of CD44 + It can regulate the role of tumor-initiating cells in self-renewal, sphere formation, and drug resistance potential. Meanwhile, in AML, the JAK / STAT (STAT4) pathway may interact with CD44 and indirectly regulate PIM2.

[0013] The role of CD44 in activating the kinase compensation pathway to restore suppressed BAD phosphorylation after TKI treatment Figure 17A shows CD33 after TKI treatment. + CD44 + pBAD + This is an explanatory diagram of the survival mechanism of blasts. In the untreated state, there is an intracellular homeostatic balance scale (BCL-2 vs. BAX) inside AML blasts. After 3 days of treatment with QUIZ or GILT, non-phosphorylated BAD accumulated. Non-phosphorylated BAD tips the scale toward cell death. Over the next few days, TKI-resistant blasts (CD33 + CD44 + pBAD +A compensatory pathway was activated in CD33 cells, resulting in phosphorylated BAD (pBAD), which compensated for the loss of PIM1 due to TKI inhibition (details of the intracellular mechanism are shown in Figure 17B). + CD44 + pBAD + Illustration of the intracellular changes in blast cells. The presence of pBAD tips the scales in favor of cell survival.

[0014] For example, tumorigenic clusters were formed 10 days after QUIZ or GILT treatment when observed under a microscope. These tumorigenic clusters were collected for FACS staining and were identified as CD33 + CD44 + pBAD + In this time interval, PIM2, PIM3, TYK2, STAT4, SOCS1, SOCS2, SHP2, and PIAS2 mRNAs achieved their peak fold-change values ​​compared to untreated controls (detailed in Figure 4E). During the next two weeks, many floating tumorigenic clusters were found, which gradually became confluent as floating single blasts. During this time, PIM2, PIM3, TYK2, STAT4, SOCS1, SOCS2, SHP2, and PIAS2 mRNAs were downregulated and reached intracellular homeostasis.

[0015] Figure 17B shows CD33 after TKI treatment. + CD44 + pBAD +Schematic diagram of intracellular changes in blasts. TKI treatment blocks FLT3-related downstream signaling, preventing the STAT5-PIM1 pathway from phosphorylating BAD. Unphosphorylated BAD then binds to BCL-2, inhibiting the anti-apoptotic role of BCL-2, resulting in a large number of blast deaths. Meanwhile, to survive TKI treatment, TKI-resistant blasts activate compensatory pathways with cytokines released from dead blasts / debris. The activated JAK / STAT signaling pathway or CD44 pathway then stimulates gene expression of alternative kinases, such as PIM2 / PIM3, family genes of PIM1, to restore phosphorylation of BAD. pBAD loses its binding function with BCL-2, preventing the initiation of apoptosis and promoting the survival of TKI-resistant blasts.

[0016] To restore intracellular homeostasis within TKI-resistant blasts, increased CD33 recruits more SHP1 / SHP2 to dephosphorylate pBAD into functional BAD. Furthermore, STAT4 activates the intracellular inhibitory pathway SOCS1 / SOCS3 and suppresses SHP1 / 2, CD33, other activated kinases, and itself, thereby preventing overactivated compensatory pathways and maintaining intracellular homeostasis in TKI-resistant blasts.

[0017] CD44 is a surface receptor on many cells, including HSCs and AML blasts, that interacts with ligands such as hyaluronan. In breast cancer, CD44 + Among the populations, highly tumorigenic stem cells were found. Increased expression of CD44 (e.g., CD44high) plays a key role in increasing adhesion of malignant HSCs to bone marrow (BM) matrix, impairing normal differentiation and accumulation of immature blasts in the BM niche. This supports previous studies showing that CD44 expression levels are higher in myelodysplastic syndrome patients progressing to AML compared to healthy controls. Examples herein show that TKI-resistant blasts are initially expressed as pBAD +The data show that TKI-resistant blasts express high CD44 along with TKI-specific activators. Time-course studies showed that the increased expression of CD44high and pBAD in resistant blasts gradually decreased as they regained their ability to survive, cluster, and proliferate confluently. Mechanistically, CD44 and its variant isoforms are known to activate different downstream signaling pathways, including PI3K / ACT and Src / MAPK, leading to cancer cell invasion and proliferation. Blocking hyaluronan, the major ligand of CD44, inhibited phosphorylation of BAD. qPCR analysis of different kinases disclosed herein also showed a significant increase in mRNAs of PIM2, PIM3, AKT1, etc. in TKI-resistant blasts. The data indicate that after TKI treatment, increased CD44 leads to activation of the PIM2 / 3 kinase compensation pathway. TKI-suppressed PIM1 kinase activity is restored, phosphorylating BAD protein and preventing its inhibitory role in BCL-2 (Figure 17B). In hepatocellular carcinoma, the JAK / STAT (STAT3) pathway is involved in the regulation of CD44 + It has been recently reported that STAT4 regulates the self-renewal, sphere formation, and drug resistance potential of tumor-initiating cells. However, our data herein showed that the expression of STAT4 was increased after TKI treatment, and PIM2 was found to be one of the STAT4 target genes. Therefore, the JAK / STAT (STAT4) pathway may interact with CD44 and indirectly regulate PIM2 in AML (Figure 17B).

[0018] CD33 is a well-known marker on the surface of AML blasts and is involved in their proliferation and survival, but the exact role of CD33 and its regulation remain to be elucidated. It has been reported that in pediatric AML, CD33 expression on the surface of blasts is reduced and correlates with a favorable prognosis. Previously, it was reported that CD33 is a myeloid-specific inhibitory receptor that contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and functions to recruit the phosphorylation enzymes SHP-1 and SHP-2. During inflammation, cytokine-induced SOCS3 not only binds to the ITIM of CD33 to compete with SHP1 / SHP2 but also promotes proteasomal degradation of itself and CD33. In addition, both SOCS1 / SOCS3 are target genes of STAT4. In this disclosure, FACS data showed that the JAK / STAT (STAT4) pathway and CD33 expression increased in TKI-resistant blasts, gradually decreasing the levels of controls, consistent with the qPCR data that the mRNAs of SOCS1, SOCS3, SHP1, SHP2, and PIAS2 were significantly increased, followed by downregulation of these mRNAs after blast recovery. The JAK / STAT (STAT4) pathway is involved in initiating an inhibitory pathway that suppresses the hyperresponsiveness after TKI treatment in resistant blasts, while CD33 is involved in recruiting SHP1 / SHP2 for dephosphorylation of pBAD to balance survival and cell death (Figure 17B). The up-down changes of CD33 and CD44 in TKI-resistant blasts were utilized to select immunotherapies such as bispecific monoclonal antibodies, chimeric antigen receptor T cell therapy (CAR-T), or bioengineered tumor infiltrating lymphocytes (TIL) that target CD33 and / or CD44 for the treatment of AML.

[0019] In some embodiments, the method of the present disclosure inhibits TKI-activated NFκB2-MIF / CXCL-CXCR2 compensation pathway. After TKI treatment, cytotoxicity-induced injury signals can directly activate the non-canonical NFκB2 (P100 / P52) pathway to release more MIF, CXCL5, CXCL8, and other tumor-promoting inflammatory cytokines. In addition to blocking immune cells, MIF acts as an autocrine signal to initiate survival mechanisms through the MIF-CD74 / CD44 pathway. Meanwhile, MIF-CXCR2, CXCL5-CXCR2, and CXCL8-CXCR2 pathways can be involved in cell proliferation by activating the G1 to S phase transition of CDK4 / CYCLIN E-based cell cycle progression. In addition to its role in pBAD-based survival (Figure 17B), TKI activation TYK2-STAT4-PIM2 / 3 may phosphorylate STAT4 (pSTAT4), thereby regulating NFκB2-MIF-CXCR2 and allowing relapse / proliferation of viable blasts (Figure 17C). Figure 17C is an illustration of TKI-activated pathways involved in blast survival and proliferation. Therefore, inhibiting tumor-promoting inflammation is also important to prevent AML relapse.

[0020] In some aspects, the disclosure provides methods for detecting TKI-resistant AML blasts in a subject having or at risk of having AML by determining the levels of one or more biomarkers CD33, CD44, and phosphorylated BAD (pBAD). In some embodiments, CD33-positive, CD44-positive, and pBAD-positive (CD33 + CD44 + pBAD + ) are determined to be at increased risk of developing TKI resistance. + CD44high + pBAD + The AML blasts are determined to be at increased risk of developing TKI resistance. CD33 is also known as Siglec-3, sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, or p67. The UniProt ID for human CD33 is P20138. CD44 is also known as CDW44, CSPG8, ECMR-III, HCELL, HUTCH-I, IN, LHR, MC56, MDU2, MDU3, MIC4, or Pgp1. The UniProt ID for human CD44 is P16070. Bcl-2-associated agonist of cell death (BAD) is also known as BBC2 and BCL2L8. The UniProt ID for human BAD is Q92934. As used herein, "CD44high", "CD44high + " or "CD44 hi " is CD44 + For example, the boxes in the third and fourth panels of Figure 2C indicate CD44high + "CD44low", "CD44low + " or "CD44 lo " is CD44 + Refers to low expression of CD44 in a population. + Cells can be classified into "CD44high" and "CD44low" subpopulations by methods known in the art, such as flow cytometry (e.g., Chaffer et al. 2013 Cell 154:61-74; Ghuwalewala et al. 2016 Stem Cell Res 16:405-417).

[0021] Evaluating subjects at high risk of developing TKI-resistant AML according to the methods of the present disclosure allows for the prescription of targeted therapy for such subjects. In some embodiments, the targeted therapy comprises a TKI that is an FLT3 inhibitor, such as midostaurin (MIDO), sorafenib (SORA), gilteritinib (GILT), and QUIZ (QUIZ).

[0022] Disclosed herein is a method of treating acute myeloid leukemia (AML) in a subject, comprising administering to the subject a therapeutically effective amount of a TKI and a therapeutically effective amount of an inhibitor of a TKI-activated compensatory pathway or an inhibitor of intracellular homeostasis necessary for leukemia relapse after treatment. In some embodiments, the subject has TKI-resistant AML or is at risk of developing TKI-resistant AML. The TKI of the combination therapy can be one or more of MIDO, SORA, GILT, and QUIZ. The inhibitor of a TKI-activated compensatory pathway can be an inhibitor of the JAK-STAT pathway, the TYK2-STAT4-PIM2 / PIM3 pathway, and / or the NFκB2 (P100 / P52)-MIF-CXCR2 pathway. In some embodiments, the inhibitor can be one or more of a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor. In some particular embodiments, the method comprises administering to the subject a therapeutically effective amount of a TKI, a therapeutically effective amount of a BCL2 inhibitor, and a therapeutically effective amount of an NFκB inhibitor. In other embodiments, the method includes administering to the subject a therapeutically effective amount of a TKI, a therapeutically effective amount of a STAT4 inhibitor, a therapeutically effective amount of a BCL2 inhibitor, and a therapeutically effective amount of an NFκB inhibitor. In some embodiments, the STAT4 inhibitor is (R)-lisofylline. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the NFκB inhibitor is MG-132 or BAY11-7082. In some embodiments, the CD44 inhibitor is Angstrom6, a urokinase-derived peptide. In some embodiments, the CXCR2 inhibitor may be a potent and selective non-peptide antagonist, such as SB225002. Examples of combinations of a TKI with an inhibitor of a TKI-activated compensatory pathway are shown in Tables 1 and 2.

[0023] In some embodiments, administration of a combination of a TKI (e.g., GILT) and an inhibitor of a TKI-activated compensatory pathway (such as a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor) synergistically increases cell death of AML tumor cells compared to administration of a TKI alone or an inhibitor of a TKI-activated pathway alone. In some embodiments, administration of a combination of a TKI (e.g., GILT) and an inhibitor of a TKI-activated compensatory pathway (such as a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor) synergistically increases cell death of certain AML tumor cells, such as Ki67 inhibitors, compared to administration of a TKI alone or an inhibitor of a TKI-activated pathway alone. + CD33 + CD44 + pBAD + After the cells are detected, they are administered to the patient.

[0024] Administration of a combination of a therapeutically effective amount of a TKI and a therapeutically effective amount of an inhibitor of a TKI activation compensatory pathway has a therapeutically effective effect on the progression of the disease compared to administration of a TKI alone or an inhibitor of a TKI activation pathway alone, and is preferably about 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 20 to 90%, 30 to 90%, 40 to 90%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 20 to 90%, 30 to 90%, 40 to 90%, 50 to 100%, 70 to 100%, 80 to 100%, 1 ... ~90%, 60~90%, 70~90%, 100~1000%, 200~1000%, 300~1000%, 400~1000%, 500~1000%, 600~1000%, 700~1000%, 800~1000%, 200~900%, 300~900%, 400~900%, 500~900%, 600~900%, 700~900%, or over 1000% (For example, about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or 1000%. In some embodiments, the method can increase cell death of AML tumor cells by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more.

[0025] In some embodiments, administering a combination of a therapeutically effective amount of a TKI and a therapeutically effective amount of an inhibitor of a TKI-activated compensatory pathway (e.g., a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor) synergistically reduces or inhibits AML blast relapse compared to administering a TKI alone or an inhibitor of a TKI-activated pathway alone. Administering a combination of a therapeutically effective amount of a TKI and a therapeutically effective amount of an inhibitor of a TKI-activated compensatory pathway ... by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, or 70% or more compared to administering a TKI alone or an inhibitor of a TKI-activated pathway alone. The AML blast relapse can be reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, for example, by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0026] Exemplary combinations of TKI and one or more inhibitors of TKI-activated compensation pathway are shown in Table 2 below with "X". Each inhibitor of TKI-activated compensation pathway includes its species and variants such as drugs with inhibitory activity. Combinations are not limited to those listed herein. Any other drug and drug combinations may be used in the method according to the present disclosure.

[0027] TIFF2024545904000002.tif214170 TIFF2024545904000003.tif203170 TIFF2024545904000004.tif203170 TIFF2024545904000005.tif177170 EXAMPLES

[0028] The following examples are offered by way of illustration and not by way of limitation.

[0029] Example 1 Biomarkers for TKI-resistant AML blasts AML cells were analyzed at different time points (3, 10, 20, and 28 days) after TKI exposure. First, MV4-11 and MOLM-14 cells were treated with TKIs in vitro using MIDO, SORA, GILT, and QUIZ. After 3 days, FACS of surface markers and intracellular proteins, including CD14, CD33, CD44, and Ki67, was performed. Figures 1A-1E show the therapeutic effects of different TKIs on MV4-11 and MOLM-14 cells in vitro (N=6). Figure 1A shows representative FACS (fluorescence-activated single-cell sorting) plots for MV4-11 cells after 3 days of in vitro treatment with different TKI inhibitors and 5-azacytidine (AZA) and 1α,25-dihydroxyvitamin D3 (VD3). Figure 1B shows representative FACS plots for MOLM-14 cells after 3 days of in vitro treatment with different TKI inhibitors and AZA / VD3. The circles indicate the clusters of viable blasts located on the FACS plot, and the green arrows indicate the tendency of blasts to die as they become viability dye positive over the next few days. Figures 1C and 1D are graphical representations of the cumulative FACS percentage data of viable MV4-11 and MOLM-14 cells. * P<0.05. Figure 1E shows representative FACS plots of Ki67 expression in different treatment groups after 3 days of in vitro treatment. Black arrows indicate Ki67 expression curves and treatment groups with lower Ki67 expression compared to untreated controls.

[0030] All four TKIs significantly reduced viable MV4-11 and MOLM-14 blasts compared to untreated controls (Figure 1A-D). +In contrast to the large population of cells, these TKIs acted mainly through cytotoxicity rather than inducing blast maturation (FACS plots for AZA and VD3, Fig. 1A,B). Among the TKIs, the second-generation therapeutics GILT and QUIZ were more effective than the first-generation FLT3 inhibitors MIDO and SORA, as expected (Fig. 1A-D). Moreover, all four TKIs significantly reduced the proliferation of viable blasts compared to untreated controls (Ki67 histograms, Fig. 1E). Morphologically, newly formed clusters of blasts were observed in MIDO- and SORA-treated cultures, but not in GILT- and QUIZ-treated cells. The remaining blasts showed a continuous tendency to apoptosis several days after treatment (green arrows, Fig. 1A,B).

[0031] To follow the cell fate of TKI-treated MV4-11 and MOLM-14, the blasts were continued to be cultured with medium changes every 2–3 days. After 10 days of TKI treatment, both MIDO- and SORA-treated MV4-11 and MOLM-14 were found to have grown confluently in culture and had normal morphology similar to untreated controls (MIDO, SORA images, Fig. 2A). On the other hand, in GILT-treated cultures, there were many newly formed tumorigenic clusters among many floating dead cells, and in QUIZ-treated cultures, there were few newly formed tumorigenic clusters (arrows, GILT, QUIZ images, Fig. 2A). The BAD protein is a proapoptotic member of the BCL-2 gene family involved in the initiation of apoptosis. Dephosphorylated BAD is proapoptotic by binding to and inactivating BCL-2, whereas phosphorylated BAD (pBAD) is antiapoptotic by liberating BCL-2 and inhibiting BAX-triggered apoptosis. FACS data showed a significant increase in pBAD expression at an early stage (3 days after treatment) in both MV4-11 and MOLM-14 treated with MIDO and SORA (pBAD histograms, FIG. 2B). At 10 days after TKI treatment, FACS analysis showed that viable CD44+ cells were significantly increased in GILT (28.7%) and QUIZ (93.1%) treated cultures in contrast to the control (0.093%), MIDO (0.2%) and SORA (0.24%) experimental groups. + pBAD + The population of viable Ki67 cells was significantly increased in GILT (17.4%) and QUIZ (68.2%)-treated cultures in contrast to control (2.32%), MIDO (2.54%), and SORA (2.54%)-treated cultures. + CD33 + CD44 + The population of cells was also significantly increased (arrows, Fig. 2E, F).

[0032] At 20 days after TKI treatment, GILT-treated MV4-11 grew confluently in culture without forming tumorigenic clusters and displayed normal morphology similar to untreated controls (Fig. 3A). However, in Quiz-treated cultures, there were still many tumorigenic clusters present among floating viable cells (arrows, image of QUIZ, Fig. 3A). FACS analysis revealed that viable CD44 + pBAD + The data revealed that the population of cells was reduced to levels (1.1%) similar to those in control (1.67%), MIDO (0.43%) and SORA (0.8%) treated cultures (arrows, Fig. 3B,C). The data also revealed that the previously significantly increased viable Ki67 expression level in GILT treated cells (17.4%) (arrow, Fig. 2E). + CD33 + CD44 + The results showed that the population of viable CD44 cells was reduced to a similar level (1.27%) as in the control (2.54%), MIDO (1.41%) and SORA (1.62%) treated cultures (Fig. 3D,E). Similar findings were observed in the QUIZ treated group, with the previously significantly increased viable CD44 cells in QUIZ (93.1%) (arrow, Fig. 2C). + pBAD + The population of viable Ki67 cells was reduced to 20.7% (arrows, Fig. 3B,C) and was significantly increased in QUIZ (68.2%) (red arrow, Fig. 2E). + CD33 + CD44 + The population of TKI-resistant MV4-11 cells was reduced to 52.8% (arrows, Fig. 3D, E), consistent with the current recovery state with many tumorigenic clusters. In summary, this data indicates that TKI-resistant MV4-11 cells express CD33 + CD44 + pBAD + Cells, Ki67 + Further time course studies showed that the increased expression of CD44, pBAD, and CD33 in resistant blasts gradually decreased as they regained the ability to survive, cluster, and grow confluently.

[0033] Example 2 TKI-activated cell survival mechanism: TYK2-STAT4-PIM2 / 3 pathway Expression of pro-survival genes in TKI-treated leukemia cells was examined at different time points after TKI exposure (days 3, 10, 20, and 28).Data indicate intracellular homeostasis changes (stage 1 activation, stage 2 cool-down) within TKI-resistant blasts.

[0034] MV4-11 cells (3 days after TKI treatment) were harvested and analyzed by quantitative reverse transcription polymerase chain reaction (qPCR). The qPCR data of four genetic aspects, including cell death, kinases specialized for phosphorylation of BAD, JAK / STAT signaling pathway, and intrinsic inhibitory pathway, were compared. The data showed that cell death-related BAX mRNA, BCL-2 mRNA, and BAD mRNA were significantly increased by 68-fold, 49-fold, and 23-fold, respectively, in GILT-treated MV4-11 cells compared to control untreated cells (Figure 4A). These findings were consistent with the increase in the number of dead cells (Viability Dye) in GILT-treated MV4-11 cells. +This is consistent with the FACS data showing that BAX mRNA, BCL-2 mRNA, and BAD mRNA were significantly increased by 44-fold, 31-fold, and 16-fold, respectively, in QUIZ-treated MV4-11 cells compared to control untreated cells (Figure 4A). Next, to investigate which kinases are involved in the phosphorylation of BAD (pBAD) after TKI treatment, we screened kinase candidates that have been reported to phosphorylate BAD under physiological conditions. In contrast to the significant decrease in PIM1 mRNA in TKI-treated cells compared to control untreated cells, PIM2 mRNA and PIM3 mRNA in GILT-treated MV4-11 cells were significantly increased compared to controls (Figure 4B), consistent with previous reports of PIM2-associated TKI resistance. For example, qPCR data showed that the fold increase of PIM2 and PIM3 mRNA in GILT-treated MV4-11 cells was the highest among all kinase genes screened (203-fold increase for PIM2 and 134-fold increase for PIM3, compared with 10-fold increase for AKT1 and 15-fold increase for PRKACA, Figure 4B). Similar expression changes were observed in QUIZ-treated MV4-11 cells compared to control untreated cells, with a significant 0.5-fold decrease, 120-fold increase, and 81-fold increase, respectively (Figures 4B and 4C). Elevated gene expression of both PIM2 and PIM3 has been found to be associated with reduced survival in adult AML patients. Overexpressed PIM3 has also been found to promote proliferation of AML cells and protect them from spontaneous apoptosis by phosphorylating BAD.

[0035] The Janus kinase (JAK) / signal transduction and activator of transcription (STAT) pathway, which consists of cytokine receptors and transmembrane proteins that recognize specific cytokines, is important in healthy blood formation and immune response. However, dysregulation of the JAK / STAT signaling pathway confers a survival advantage to tumorigenic cells by transmitting anti-apoptotic and proliferative signals in various cancers, including hematological malignancies. In FLT3 in AML, a significant increase in STAT5 from the JAK / STAT pathway is associated with sustained activation of the FLT3 signaling pathway, resulting in AML cell survival and proliferation. With this in mind, we performed a screen of total gene expression of the JAK / STAT pathway. The data showed that in GILT-treated MV4-11 cells, TYK2 mRNA and JAK2 mRNA were significantly increased by 71-fold and 6-fold, respectively, compared to control untreated cells (Figure 4D). A similar pattern of changes in the expression of TYK2 and JAK2 mRNA was observed in MV4-11 cells treated with Quiz compared to control untreated cells, with a significant increase of 46-fold and 6-fold, respectively (Figure 4D). Furthermore, the fold increase of both STAT4 and STAT2 mRNA in GILT-treated MV4-11 cells compared to control untreated cells was the highest among all STAT genes screened (23-fold increase in STAT4 and 9-fold increase in STAT2, compared to 4-fold increase in STAT5A and 2-fold increase in STAT3, Figure 4D). Similar findings were observed for STAT4 and STAT2 mRNA in MV4-11 cells treated with Quiz compared to control untreated cells, with a significant increase of 19-fold and 6-fold, respectively (Figure 4D). This significant increase in STAT4 is consistent with a recent report that there are STAT4-mediated (TKI-resistant) and STAT5-mediated (TKI-sensitive) pathways that can regulate the role of PIM2 in BAD phosphorylation in chronic myeloid leukemia (CML). STAT4 activation involves the recruitment of STAT2.

[0036] We next investigated how the intrinsic homeostasis of blasts is switched off, particularly cytokine signaling and kinase activation. Signaling through the JAK-STAT pathway is tightly regulated by many endogenous feedback inhibitors, including suppressor of cytokine signaling (SOCS), inhibitor of activated STAT proteins (PIAS), and protein tyrosine kinases (PTPs) such as SHP1 / 2. Therefore, we performed a screen for gene expression of the SOCS family. Data showed that SOCS1 mRNA and SOCS3 mRNA were significantly increased by 153- and 182-fold, respectively, in MV4-11 cells treated with GILT compared to control untreated cells (Figure 4E). Similar findings were observed in MV4-11 cells treated with Quiz compared to control untreated cells, with the mRNA significantly increased by 88- and 120-fold, respectively (Figure 4E). Furthermore, genes encoding kinases such as SHP1 mRNA and SHP2 mRNA in GILT-treated MV4-11 cells were significantly increased when compared to those in control untreated cells (SHP2 increased 9-fold, SHP1 increased 4-fold, and PIAS2 increased 2-fold, Fig. 4E). PIAS family member, inhibitor of activated STAT2 protein (PIAS2), has been reported to be a negative regulator of Stat4. Similar fold increases were observed in SHP1 mRNA and SHP2 mRNA in Quiz-treated MV4-11 cells compared to control untreated cells (SHP2 increased 5-fold, SHP1 increased 3-fold, and PIAS2 increased 2-fold, Fig. 4E).

[0037] Ten days after TKI treatment, the mRNAs of PIM2 (355-fold increase), PIM3 (494-fold increase), TYK2 (73-fold increase), STAT2 (14-fold increase), STAT4 (28-fold increase), SOCS1 (615-fold increase), SOCS3 (590-fold increase), SHP2 (9-fold increase), and PIAS2 (570-fold increase) reached their peak fold changes in QUIZ-resistant blasts compared to untreated controls (Figures 4F-4I). Increases in the mRNAs of these genes were similarly observed 20 days after TKI treatment (Figures 4J-4M). At this time point, the majority of surviving QUIZ-resistant blasts were CD44 + pBAD + (93.1%, red arrow, Fig. 2C), which had begun to form a small number of tumorigenic clusters (arrow, Fig. 2A). In summary, the qPCR and FACS data indicate that the TYK2-STAT2 / 4-PIM2 / 3 pathway plays a critical role in the survival and tumorigenic growth of TKI-resistant blasts. Meanwhile, the upregulated corresponding inhibitory pathways suggest the existence of an endogenous homeostatic system that suppresses the overactivation of the JAK / STAT pathway and supports the recovery of TKI-resistant blasts.

[0038] We investigated late TKI-resistant blasts after TKI treatment. At 28 days after TKI treatment, QUIZ-treated MV4-11 cells grew confluently in culture without forming tumorigenic clusters and showed normal morphology similar to untreated controls (not shown, but similar to images of GILT, Fig. 3A). FACS analysis revealed that viable CD44 + pBAD + We found that the population of cells had decreased to levels (0.24%) similar to those in control (1.17%), MIDO (1.23%), and SORA (1.05%) treated cultures (arrows, Figures 5A and 5C). We also found that the survival Ki67 expression level, which had previously been significantly increased in QUIZ (52.8%) (arrow, Figure 3D), was reduced. + CD33 + CD44 +The population of cells was reduced to levels (1.58%) similar to those in control (2.6%), MIDO (2.54%), and SORA (2.82%) treated cultures (arrows, Figures 5B,D).

[0039] After 28 days, all four TKI-treated blasts relapsed in vitro. We also assessed whether gene expression was restored. The same qPCR primer sets were used on RNA isolated from blasts after 28 days of treatment to compare four genetic aspects, including cell death, kinases specific to phosphorylation of BAD, the JAK / STAT signaling pathway, and the intrinsic inhibitory pathway. The data showed that BAX mRNA, BCL-2 mRNA, and BAD mRNA were not significantly different in MV4-11 cells treated with TKIs compared to those in control untreated cells (Figure 6A). Among the kinase genes, a significant decrease in PIM1 mRNA was observed in all TKI-treated MV4-11 cells (Figure 6B). Meanwhile, only QUIZ-treated MV4-11 cells showed a significant increase in PIM2 and PIM3 mRNA by approximately 2-fold (Figure 6B). There was no significant difference in JAK / STAT in MV4-11 cells treated with TKIs compared to those in control untreated cells (Figure 6C). Among the inhibited pathways, only QUIZ-treated MV4-11 cells still showed significant increases in CISH mRNA, SOCS1 mRNA, and SOCS3 mRNA, approximately 2-fold, compared to those of control untreated cells (Figure 6D). In summary, the data for late TKI-resistant blasts are consistent with the confluent relapse of blasts in vitro, as late TKI-resistant blasts are more similar to untreated cells than early TKI-resistant blasts.

[0040] The time course studies shown in Examples 1-2 demonstrated that after TKI treatment, recurrent tumorigenic blasts express CD33 + CD44 + pBAD + cells, demonstrating that they are undergoing intracellular homeostasis to regenerate leukemic proliferation (Figure 17A).

[0041] Example 3 Release of macrophage migration inhibitory factor (MIF) and upregulation of CXCR2 by TKI-treated blast cells To identify proteins involved in the initiation of survival mechanisms and subsequent proliferation of AML blasts, human cytokine proteome profile arrays were performed on cell-free supernatants of cultures of MV4-11 treated with GILT. Remarkably, the data revealed that macrophage migration inhibitory factor (MIF) was significantly increased by 2.6-fold in the supernatants of MV4-11 after treatment compared to untreated controls (arrows, Figure 7A; average pixel density is shown in Figure 7B). Under no treatment (No Tx) conditions, MV4-11 releases numerous cytokines such as MIF in its supernatant, but one dose of GILT treatment abolished the release of various cytokines such as osteopontin (arrow, Figure 7A), a protein closely associated with poor prognosis in AML patients. Next, bone marrow mononuclear cells (BMMNCs) from AML patients were treated ex vivo for 3 days. In GILT-treated AML samples (newly diagnosed patient #1 and refractory patient #2, Table 4), MIF release was also found to be significantly increased by 2.1-fold in the supernatant compared to untreated controls (Figures 7C, 7D).

[0042] To investigate which transmembrane proteins interact with MIF to signal after different TKI treatments, we compared gene expression of all encoded MIF receptors. qPCR data showed that CXCR2 mRNA was significantly increased (109-fold) in MV4-11 cells treated with GILT (Figure 7E). The increase in fold change of CXCR2 mRNA was the highest among all MIF receptors screened (Figure 7E). CXCR1 mRNA also showed a high increase (about 90-fold increase, not shown). The data are consistent with a recent report that high expression of CXCR2 correlates with poor prognosis in AML patients. In summary, the data reveal that 1) both AML cell lines and primary AML BMMNCs release more MIF after TKI treatment and 2) TKIs activate a marked upregulation of CXCR2 gene expression in MV4-11 cells.

[0043] Example 4 MIF promotes the proliferation of MV4-11 through the MIF / CXCL-CXCR2 pathway and increases the CD44High expression after TKI treatment. + Promotes survival of blast cells Significantly increased MIF expression has been associated with angiogenesis, cell cycle initiation, and tumor metastasis in solid tumors, but the exact role of MIF in AML has not yet been fully elucidated. To investigate the role of MIF in the proliferation of AML blasts, cell cultures of MV4-11 (an AML blast cell line) were supplemented with different doses of recombinant human MIF peptide. FACS data showed that the lowest dose, 5 ng / ml MIF, significantly increased the Ki67 +We showed that MIF could induce MV4-11 proliferation by expressing a higher percentage of CXCR2 in MV4-11 cells (Figure 8A). To investigate how MIF affects immune cells, MIF was tested in RAW264.7 (a macrophage cell line). 5ng / ml of MIF effectively suppressed RAW264.7 proliferation (Figure 8B). Furthermore, qPCR data also showed that exogenous recombinant MIF could effectively induce gene expression of chemokine (C-X-C motif) ligand (CXCL)1, CXCL5, CXCL8 (IL-8), cyclin-dependent kinase 4 (CDK4), and CYCLIN E1 in MV4-11 cells (Figure 8F-8G). This finding is consistent with previous reports that primary AML blasts constitutively express MIF and stimulate bone marrow mesenchymal cells to release CXCL8 (IL-8), a ligand that also binds to CXCR2, to sustain blast proliferation. Furthermore, CXCL1 / 5 / 8 were routinely released at high concentrations (median levels >1000 pg / mL) in the supernatants of ex vivo cultures of naive AML patient cells. Data reveal that 1) MIF induces MV4-11 proliferation, 2) MIF induces gene expression of CXCR2, the major receptor for various cytokines, 3) MIF induces gene expression of key cytokines promoting AML, 4) MIF induces gene expression of key regulators important for G1-S phase progression of the cell cycle, and 5) MIF inhibits macrophage proliferation, which may transiently block immune cells to avoid further damage. Data suggest that the MIF / CXCL / CXCR2 pathway is activated by TKI treatment to initiate tumor-promoting inflammation and support the proliferation of surviving blasts.

[0044] To investigate whether the MIF / CXCL-CXCR2 pathway can be activated in surviving blasts by TKI treatment to support their survival and continued proliferation, we performed sequential co-cultures to examine whether supplementation with exogenous MIF early (simultaneous) or late (sequential) after TKI treatment could rescue GILT-treated MV4-11 in vitro. CD44, a multifunctional cell surface adhesion receptor, has been identified as a master regulator of quiescent AML leukemia stem cells (LSCs) that are highly microenvironment-dependent. Supplementation of GILT-treated MV4-11 cells with MIF in sequential co-cultures (procedures detailed in Methods) rescued survival and continued proliferation of quiescent CD44 + The CD44 cell population was significantly increased (Fig. 8C, plot). + The percentage of the population was found to be significantly increased from 3.66% in the untreated control to 17.7% in GILT alone, 26.3% in GILT + 50 ng / mLMIF, and 25.2% in GILT + 200 ng / mLMIF (Figures 8C-8E). + and Ki67 - CD44High, which includes both subsets + Another group of cells was also found (Figure 8F). + The cell population could be visualized by flow cytometry histogram (Figure 8G). Meanwhile, qPCR data revealed that supplementation of GILT-treated MV4-11 cells with MIF significantly increased the gene expression of CXCR2 (bar graph, Figure 8G). CD44high + Stem-like cells have previously been found to be highly tumorigenic, treatment-resistant, and responsible for metastatic progression in breast cancer. Recently, a combined genetic and functional approach to trace the origin of AML relapse has demonstrated that therapy-resistant cells are already present at diagnosis and evolve at relapse from either rare LSCs or blasts with stemness transcriptional characteristics. Our findings suggest that MIF, which was significantly increased after TKI treatment, is a key regulator of inducible CD44high + These results suggest that IL-1 expression may support pre-existing LSC-like blast populations that survive TKI treatment and lead to relapse of FLT3mut AML.

[0045] Example 5 Mechanism of TKI activation cell proliferation: NFκB-MIF-CXCR2 pathway To investigate other pathways that transduce MIF signals, we compared the gene expression of all encoded MIF receptors. qPCR data showed that CXCR2 mRNA was significantly increased (109-fold) in GILT-treated MV4-11 cells (Figure 9A). The fold increase of CXCR2 mRNA was the highest among all MIF receptors screened (Figure 9A). Furthermore, exogenous recombinant MIF effectively induced CXCR2 expression in MV4-11 blasts in vitro (Figure 9B), suggesting that MIF-CXCR2 signaling may be a proliferation pathway in TKI-resistant blasts. Another question is to identify the transcription factor that regulates the gene expression of MIF. The nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) family of transcription factors is known to control the expression of both cytokines and their receptors. The NFκB activation pathway includes two pathways: NFκB1 (p50-p65, canonical) and NFκB2 (p52-RelB, non-canonical). To investigate which NFκB pathway is responsible for TKI resistance, gene expression of the NFκB family was compared. qPCR data showed that NFκB2 mRNA was significantly increased (110-fold) in MV4-11 treated with GILT when compared to the control (Figure 9C). The fold increase of NFκB2 was the highest among all NFκB members (not shown). NFκB activation has been reported to be controlled by the JAK / STAT signaling pathway in immune cells. Also, as members of the "fast acting" transcription factors, such as c-Jun and STAT, the NFκB family is a first responder to harmful cellular stimuli. Pro-tumorigenic inflammation has been considered as a potential therapeutic target for the treatment of solid cancers.

[0046] Example 6 Targeting the MIF-CXCR2 pathway in the treatment of AML We investigated whether targeting the MIF-CXCR2 signaling pathway could effectively treat AML blasts. First, we tested the combination of GILT and ribosomal protein S19 (RPS19) on MV4-11 blasts in vitro. RPS19 is a MIF inhibitor and has been reported to suppress the cisplatin-activated MIF-CD74-NFκB signaling pathway in acute kidney injury. The data showed that the combination of 80 nM GILT and 20 μg / ml RPS19 had no synergistic anti-leukemic effect (Figure 10). We examined the combination of 80 nM GILT with 50 μM SB225002, a CXCR2 inhibitor (CXCR2-I), on MV4-11 blasts in vitro. The data showed that the combination of GILT and CXCR2-I suppressed survival CD44 + The percentage of MV4-11 blasts (0.73% vs. 12.2% in GILT alone and 92.7% in untreated controls; Fig. 11A / top plot), and Ki67 + CD44 + The results showed that GILT significantly reduced the percentage of blasts (1.66% vs. 12.2% for GILT alone, 5.7% for CXCR2-I alone, and 83.1% for untreated controls, Figures 11A / lower plots and 11B / 11C bar graphs). The anti-leukemic effect of a combination of GILT and CXCR2-I on primary BMMNCs ex vivo was examined. The data showed that GILT, together with CXCR2-I, significantly reduced the viable CD33 + CD13 + The percentage of primary blasts (1.75% vs. 7.14% for GILT alone, 3.11% for CXCR2-I alone, and 24.9% for untreated controls; Fig. 11D / top plot), and Ki67 + CD13 +The results again showed that CXCL8-CXCR2-I significantly reduced the percentage of blasts (0% vs. 60% for GILT alone, 14.3% for CXCR2-I alone, and 81.3% for untreated controls, Fig. 11D / lower plots and 11E / 11F bar graphs (patients no. 2 and 3, Table 4)). However, primary AML BMMNCs from two patients did not respond well to this treatment (patients no. 1 and 4, Table 4). The data are consistent with previous reports that inhibition of the CXCL8-CXCR2 axis moderately reduces clonal expansion of MDS / AML cell lines and patient samples. There may be another cytokine pathway that compensates for the deficiency of the MIF-CXCR2 pathway.

[0047] Example 7 NFκB2 is involved in activation of the MIF / CXCL-CXCR2 pathway in TKI-resistant blast survival To abolish the leukemic effects of various cytokines and their dynamic receptors, we investigated the transcription factors regulating their gene expression in TKI-resistant blasts. Nuclear factor kappa light-chain-enhancer of activated B cells (NFκB), a family of transcription factors, is known to be the main molecular link between inflammation and the initiation / progression of carcinogenesis by controlling the expression of various tumor-promoting inflammatory cytokines and their receptors. It has also been reported that there is an NFκB binding region in the MIF promoter, which enhances the transcription of MIF. There are two NFκB activation pathways: NFκB1 (p50-p65, canonical) and NFκB2 (p52-RelB, non-canonical). To investigate which NFκB pathway is responsible for TKI resistance, we compared the gene expression of the NFκB family. qPCR data showed that NFκB2 mRNA was significantly increased (110-fold) in MV4-11 treated with GILT when compared to the untreated control (NO-TX) (right bar graph, Figure 12A). The fold increase in NFκB2 was much higher than that of NFκB1 (left bar graph, FIG. 12A). Next, we performed siRNA knockdown of NFκB2 in vitro and then examined the genetic changes following the reduction of NFκB2 mRNA (FIGS. 12B-12E). siRNA knockdown of NFκB2 reduced NFκB2 mRNA by approximately 3-fold (siRNA-NFκB2 vs. untreated control, FIG. 12B), and by 2-fold in combination with GILT (GILT + siRNA-NFκB2 vs. GILT only, FIG. 12B). Transient knockdown of NFκB2 does not affect gene expression of NFκB1 (data not shown). Furthermore, the data show that transient silencing of NFκB2 significantly reduced MIF mRNA by 1.5-fold (siRNA-NFκB2 vs. untreated control, FIG. 12C) and GILT-activated gene expression of CXCR2 and CXCL5 by 2-fold and 2.6-fold, respectively (GILT + Only siRNA-NFκB2 vs. GILT significantly decreased the expression of NFκB2 in IL-16 cells (Figure 12D, E). In summary, the data suggest that the NFκB2-MIF / CXCL-CXCR2 pathway may be involved in initiating tumor-promoting inflammation that supports the survival and cell proliferation of TKI-resistant AML blasts.

[0048] Example 8 The combination of GILT and NFκB inhibitor (NFκB-I) effectively treated primary blasts ex vivo from newly diagnosed and refractory AML patients The combination of 80 nM GILT and 50 μM BAY11-7821 (NFκB-I) was tested as a novel therapeutic approach for AML. + NFκB-I inhibits survival CD33 + CD13 + The percentage of primary blasts (0.035% vs. 35.8% for GILT alone, 0.14% for NFκB-I alone, and 51.4% for untreated controls; Fig. 13A / top plot), and Ki67 + CD33 + The results showed that GILT synergistically reduced the percentage of proliferating blasts (0% vs. 73.3% for GILT alone, 25% for NFκB-I alone, and 78.9% for untreated controls, Figures 13A / bottom plots and 13B / 13C bar graphs) (reproducible in newly diagnosed AML patient #5 and patient #6, Table 4). Surprisingly, GILT + The NFκB-I combination completely ablated the expression of the tumor-promoting inflammatory cytokines CXCL1, CXCL5 and CXCL8 in supernatants of treated AML patient specimens compared to untreated controls (FIG. 13D / blot film and 13E bar graph).

[0049] We next investigated the anti-leukemic effect of this combination on primary BMMNCs from a patient with refractory AML (patient no. 7, Table 4). + Not only did NFκB-I significantly inhibit tumorigenic clustering in primary BMMNCs (image, Fig. 14A), but CD117 + CD13 + It significantly reduced the survival rate of blast cells (Fig. 14B / upper plot), and Ki67 + CD33 + CD117 + CD13 + Mechanistically, the qPCR data showed that GILT inhibited proliferation of primary blasts (Fig. 14B / lower plot). +We showed that the combination of NFκB-I significantly reduced the gene expression of CYCLIN E1, a molecule responsible for the S phase of the cell cycle (Figure 14C), and CD44, an adhesion molecule for tumorigenic clustering (Figure 14D). Finally, the combination treatment was found to effectively remove CXCL8 (IL-8) in the supernatants of treated AML patient specimens compared to GILT alone (Figure 15). In summary, the ex vivo data demonstrated that targeting the NFκB2-MIF / CXCL-CXCR2 pathway can effectively suppress the release of tumor-promoting inflammatory cytokines and their mediated survival and proliferation of primary blasts from both newly diagnosed and refractory AML patients ex vivo.

[0050] Example 9 TKI-based combination therapy for AML relapse in vitro The combination of TKIs and inhibitors targeting the TKI-activated compensatory system has proven effective in the treatment of refractory AML ex vivo. FLT3, the most frequently mutated gene in AML patients, is often associated with poor overall survival with an increased risk of relapse. However, the antileukemic effect of the latest FLT3 inhibitors in monotherapy or in combination with standard treatment is limited by a short duration and high relapse rate. Therefore, the question is whether targeting compensatory pathways together with the administration of FLT3 inhibitors, such as GILT, will prevent relapse. A list of in vitro studies combining inhibitors (commercially available products) targeting the new pathways of GILT and MV4-11 is shown in Table 1.

[0051] Table 1 lists various combination therapies targeting TKI-activated compensatory pathways of MV4-11 in vitro. JPEG2024545904000006.jpg80166

[0052] More comprehensive and non-limiting exemplary combination therapies are shown in Table 2. Table 1 shows examples of new combination therapies targeting the NFκB2-MIF-CXCR2 pathway. Primary AML blasts have been found to constitutively express MIF, which stimulates bone marrow mesenchymal cells to release interleukin-8 (CXCL8, IL-8, a ligand that binds to CXCR2) to maintain blast proliferation. Thus, targeting the receptor CXCR2 can prevent leukemia relapse through its ligands MIF and IL-8.

[0053] We tested the effect of combining TKIs with pathway-driven small molecule inhibitors to suppress relapse of TKI-resistant MV4-11 in vitro. A detailed description of the small molecule inhibitors, including doses and catalogues, is provided in Materials and Methods.

[0054] FACS analysis after 3 days of combination treatment revealed that GILT + BCL2 inhibitor (2.8%), GILT + PIM inhibitors (18.6%) and GILT + Cultures treated with the STAT4 inhibitor (18.7%) revealed a significantly reduced population of viable blasts in contrast to cultures treated with GILT alone (26.9%) (Figures 16A, 16B). FACS analysis after 7 days of combination treatment revealed that GILT + BCL2 inhibitor (6 cells) and GILT + In cultures treated with STAT4 inhibitors (40 cells), in contrast to cultures treated with GILT alone (180 cells), viable Ki67 + CD33 + CD44 + Consistent with the FACS results, the number of GILT cells was significantly decreased (Fig. 16C, 16D). + Although newly formed tumorigenic clusters were present in STAT4 inhibitor-treated cultures, these clusters appeared later and were fewer and smaller at 7 days after treatment than in untreated controls (FIG. 16E) and GILT-only treated cultures (FIGS. 16F, 16G). + BCL2 inhibitors and GILT +Both STAT4 inhibitor-treated cultures were much lighter in terms of population density than the control and GILT-only experimental cultures. + In cultures treated with BCL2 inhibitors, most of the floating cells died, consistent with the FACS results (image, FIG. 16H; FACS plot, FIG. 16C), and no new tumorigenic clusters formed after 2 weeks of treatment. The data suggest that combination therapy of TKIs (GILT) with pathway-driven small molecule inhibitors such as STAT4 inhibitors ((R)-lisofylline) or BCL-2 inhibitors (venetoclax) may be a novel therapeutic approach for treating refractory AML.

[0055] In summary, novel molecular and genetic phenotypes of TKI-resistant AML blasts and their response to survival from TKI treatment and maintenance of endogenous homeostasis after blast relapse are provided herein. Also provided herein is a novel therapeutic approach that is more effective than TKI treatment alone by combining TKIs with protein inhibitors that target specific JAK-STAT pathways. The experimental approach, biomarker up-down phenomenon, survival mechanism, and intracellular homeostasis concepts in treatment-resistant leukemic blasts provided herein may also be applied to other refractory malignancies.

[0056] Materials and Methods Human samples. AML bone marrow (BM) mononuclear cells (BMMNC) (patient numbers 1–7, Table 4) were obtained from City of Hope National Medical Center (COHNMC). All donor patients signed informed consent forms. Acquisition of samples was in accordance with a letter of assurance submitted and approved by the Department of Health and Human Services, was approved by the institutional review boards of LLUMC and COHNMC, and met all requirements of the Declaration of Helsinki.

[0057] Cell lines and cell culture. MV4-11 (ATCC CRL-9591) and MOLM-14 (DSMZACC-777) are human-derived AML blast cell lines with FLT3-ITD. AML cells (either MV4-11 or primary AML BMMNC) and RAW264.7 were cultured in RPMI-1640 medium (Hyclone, Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone) and 1% penicillin / streptomycin. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0058] Small molecule inhibitors and in vitro and ex vivo treatments. A list of inhibitors, abbreviations, manufacturers, and catalog numbers are in Table 3. MIDO and SORA are first generation FLT3 inhibitors, while GILT and QUIZ are second generation inhibitors. As single agents to treat blasts in vitro, a single dose of 80 nM of MIDO, GILT, QUIZ, or SORA was administered in 1 ml of 1 × 10 cells for each experimental group in 24-well plates. 6 The four TKIs were added to the cells. The dose of 80 nM was selected based on their respective dose-dependent cytotoxicity in previous reports. Treatment with 100 nM MIDO reduced viable MV4-11 by 60%. Both GILT and QUIZ reportedly began to suppress tumorigenic clustering and c-Kit at a dose of 80 nM. 100 nM SORA could significantly induce apoptosis and cell cycle arrest in MV4-11 after 72 h of treatment in vitro. A combination of 5 μM 5-azacytidine (AZA) and 80 nM 1α,25-dihydroxyvitamin D3 (VD3) was added to the experimental group as in the previous report. Combinations for treating blasts in vitro included one dose of 80 nM GILT and one dose of either 100 nM venetoclax (BCL2 inhibitor), 10 μM (R)-lisofylline (STAT4 inhibitor), 100 nM AZD1208 (PIM inhibitor), 500 nM PF-06826647 (TYK2 inhibitor), or 500 nM AT9283 (JAK2 / 3 inhibitor), in 1 ml of 1 × 10 cells per experimental group in 24-well plates. 6Three days after one dose of treatment, cells were either harvested for analysis by FACS and qPCR or replated in 24-well plates and continued to be cultured. At different time points, replated cells were harvested for analysis by FACS and qPCR. Medium was changed every 2-3 days.

[0059] For MIF in vitro experiments, different doses of MIF were added to 1 ml of 1 × 10 cells for each experimental group in 24-well plates. 6 MV4-11 or RAW264.7 cells were added to the cells. Two days after one dose of treatment, cells were harvested for analysis by FACS and qPCR. One dose of 80 nM GILT and one dose of the different inhibitors were added to 1 ml of 1 × 10 cells for each experimental group in 24-well plates as combinations for treating blasts in vitro or ex vivo. 6 MV4-11 cells or 0.5-1 x 10 6 Primary AML BMMNCs were added. Dose-response experiments of CXCR2 inhibitor and NFκB inhibitor with or without 80 nM GILT were performed. The current dose of either 50 μM CXCR2 inhibitor or 50 μM NFκB inhibitor was determined to be the most effective dose against MV4-11 based on FACS analysis. After 3 days of treatment with one dose, cells were harvested for analysis by FACS and qPCR.

[0060] Transfection of siRNA-NFκB2 in MV4-11. Small interfering RNA (siRNA) was used to transiently knockdown NFκB2 (p100 / 52) in MV4-11 cells. Cells transfected with scrambled siRNA were used as a control (siRNA-negative control). Cells were grown in 24-well plates and transfected with siTran2.0 siRNA transfection reagent (Origene catalog no. TT320002) with 50 nM NFκB2 (human, ID 4791) 27mer siRNA duplex (no. SR303162, Origene, Rockville, MD, USA). For experiments involving GILT treatment, one dose of 80 nM GILT was added simultaneously to MV4-11 cells with siRNA-NFκB2 in 24-well plates. qPCR experiments including confirmation of knockdown and gene alteration were performed 2 days after transfection.

[0061] Flow cytometry (FACS). Cells were harvested and the expression of cell surface biomarkers (CDs) and intracellular proteins was examined by multicolor FACS as previously described

[24] . Approximately 1 × 10 4 ~10 6 Cells were stained with various fluorescently labeled antibodies specific for the desired cell surface proteins for 30 min at 4°C. Surface stained cells were then fixed and permeabilized with the appropriate reagents (e.g., BD Pharmingen Cytofix / Cytoperm buffer) and stained with various fluorescently labeled antibodies specific for the desired intracellular proteins for 2 h at 4°C in permeabilization buffer (e.g., BD Perm / Wash buffer). Concentrations of Abs were used as recommended by the manufacturer. Finally, cells were washed twice with permeabilization buffer and twice with FACS buffer before analysis on a BD FACSAria II. Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

[0062] RNA isolation and real-time polymerase chain reaction (qPCR) analysis. MV4-11 and MOLM-14 cells were cultured in the presence of TKI drugs for 72 hours or replated after 28 days of TKI drug treatment. For RNA isolation and qPCR analysis, cells were isolated as described above. Total RNA was isolated using RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. First-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Life Technologies). qPCR was performed and analyzed using primers for known specific markers using an Applied Biosystems 7900HT Real-Time PCR machine. PCR conditions were 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 15 seconds. Relative expression levels of genes were determined using the ΔΔCt method and normalized to GAPDH.

[0063] Proteomics and blotting analysis. Three days after one dose of treatment, cell-free supernatants were collected for human cytokine profile assay. Human XL Cytokine Array Kit (Cat. No. ARY022B, R&D) was used according to the manufacturer's procedure and exposed to X-ray film (<10 min). Mean spot pixel density profiles were generated using a transmission scanner and ImageJ analysis software. Image acquisition. Phase contrast images were taken using an Olympus 1X71 inverted microscope and processed using Olympus cellSens Dimension 1.15 Imaging Software. Statistical analysis. Statistical significance was assessed by ANOVA or unpaired Student's t-test for comparison between two groups. All values ​​are presented as mean ± SEM. Results were considered significant when P value was less than 0.05.

[0064] Subjects and Reagents TIFF2024545904000007.tif157170

[0065] TIFF2024545904000008.tif236170

[0066] When ranges are disclosed herein, a range from any lower limit may be combined with any upper limit to recite a range not expressly recited, and similarly, a range from any lower limit may be combined with any other lower limit to recite a range not expressly recited, and similarly, a range from any upper limit may be combined with any other upper limit to recite a range not expressly recited. Furthermore, reference to values ​​recited in a range includes each and every value within that range, even if not expressly recited. Thus, every point or individual value may be combined with any other point or individual value, or any other lower or upper limit, to serve as its own lower or upper limit, to recite a range not expressly recited.

[0067] Other objects, features and advantages of the present disclosure will be apparent from the drawings, detailed description and examples set forth above. These drawings, detailed description and examples, while showing certain embodiments of the present disclosure, are given for illustration only and are not meant to be limiting. In further embodiments, features from a particular embodiment may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the particular embodiments described herein. Although the present disclosure includes certain aspects, embodiments, and optional features, it should be understood that modifications, improvements, or variations of such aspects, embodiments, and optional features may be employed by those skilled in the art, and such modifications, improvements, or variations are considered to be within the scope of the present disclosure.

Claims

1. 1. A method for assessing resistance of acute myeloid leukemia (AML) to tyrosine kinase inhibitor (TKI) therapy in a subject having AML, comprising: isolating AML tumor cells from a subject; and Detecting the expression of CD33, CD44, and phosphorylated BCL2-associated cell death agonist (pBAD) on the surface of AML tumor cells Including, The method, wherein the subject is determined to be at high risk of developing TKI-resistant AML in response to the AML tumor cells being CD33 positive, CD44 high positive, and pBAD positive.

2. The method of claim 1, wherein the CD33 positive, CD44 high positive, and pBAD positive tumor cells are Ki67 positive.

3. 1. A method of treating acute myeloid leukemia (AML) in a subject, comprising: administering to the subject a therapeutically effective amount of a TKI and a therapeutically effective amount of one or more inhibitors of a TKI-activated compensatory pathway, thereby treating AML in the subject. The method includes:

4. The method of claim 3, wherein the inhibitor inhibits the JAK-STAT pathway.

5. The method of claim 3 or 4, wherein the inhibitor inhibits the TYK2-STAT4-PIM2 / PIM3 pathway.

6. The method of any one of claims 3 to 5, wherein the inhibitor inhibits the NFκB2 (P100 / P52)-MIF-CXCR2 pathway.

7. The method of any one of claims 3 to 6, wherein the one or more inhibitors are one or more of a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor.

8. The method of claim 3, comprising administering to the subject a therapeutically effective amount of each of a TKI, a BCL2 inhibitor, and an NFκB inhibitor.

9. The method of claim 3, comprising administering to the subject a therapeutically effective amount of each of a TKI, a STAT4 inhibitor, a BCL2 inhibitor, and an NFκB inhibitor.

10. The method according to any one of claims 3 to 9, wherein the TKI is gilteritinib.

11. The method of any one of claims 3 to 9, wherein the TKI is quizartinib.

12. The method of claim 7 or 9, wherein the STAT4 inhibitor is (R)-lisofylline.

13. The method of any one of claims 7 to 9, wherein the BCL2 inhibitor is venetoclax.

14. The method according to any one of claims 7 to 9, wherein the NFκB inhibitor is MG-132 or BAY11-7082.

15. The method of claim 7, wherein the CD44 inhibitor is Angstrom 6.

16. 8. The method of claim 7, wherein the CXCR2 inhibitor is SB225002.

17. The method of any one of claims 3 to 16, wherein the subject has TKI-resistant AML or is at risk of developing TKI-resistant AML.

18. The method of any one of claims 3 to 17, wherein the subject has one or more mutations in the FMS-like tyrosine kinase 3 (FLT3) gene.

19. 1. A method of treating acute myeloid leukemia (AML) in a subject, comprising: Isolating AML tumor cells from a subject; Detecting the expression levels of CD33, CD33, CD44, Ki67, and phosphorylated BCL2-associated cell death agonist (pBAD) on the surface of AML tumor cells; and in response to the tumor cells being CD33 positive, CD44 high positive, Ki67 positive, and pBAD positive, administering a therapeutically effective amount of a tyrosine kinase inhibitor (TKI) and a therapeutically effective amount of one or more of a STAT4 inhibitor, a BCL2 inhibitor, a CD44 inhibitor, a CXCR2 inhibitor, and an NFκB inhibitor. The method includes:

20. 20. The method of claim 19, wherein the TKI is gilteritinib or quizartinib.

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